Recombinant infectious bovine rhinotracheitis virus

ABSTRACT

The present invention provides a recombinant infectious bovine rhinotracheitis designated S-IBR-052 (ATCC Accession No. VR 2443). The present invention also provides a vaccine which comprises an effective immunizing amount of the recombinant infectious bovine rhinotracheitis virus designated S-IBR-052 and a suitable carrier.The present invention provides homology vectors, methods of immunization and a method of distinguishing an animal vaccinated with the vaccines of the present invention from an animal infected with a naturally-occurring infectious bovine rhinotracheitis virus.

This application is a national stage application under 35 U.S.C. 371 ofPCT International Application No. PCT/US95/01491, filed Feb. 2, 1995, acontinuation-in-part of U.S. Ser. No. 08/191,866, filed Feb. 4, 1994,now U.S. Pat. No. 5,783,195, and a continuation-in-part of U.S. Ser. No.08/334,428, filed Nov. 4, 1994, now U.S. Pat. No. 5,834,305, which is acontinuation of U.S. Ser. No. 08/037,707, filed Mar. 25, 1993, nowabandoned, which is a continuation of U.S. Ser. No. 07/649,380, filedJan. 31, 1991, now abandoned, which is a continuation of U.S. Ser. No.07/078,519, filed Jul. 27, 1987, now abandoned.

Within this application several publications are referenced by arabicnumerals within parentheses. Full citations for these publications maybe found at the end of the specification immediately preceding theclaims. The disclosures of these publications are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

The ability to isolate viral DNA and clone this isolated DNA intobacterial plasmids has greatly expanded the approaches available to makeviral vaccines. The methods used to make the present invention involvemodifying cloned viral DNA sequences by insertions, deletions and singleor multiple base changes. The modified DNA is then reinserted into theviral genome to render the virus non-pathogenic. The resulting livevirus may then be used in a vaccine to elicit an immune response in ahost animal and to protect the animal against a disease.

One group of animal viruses, the herpesviruses or Herpetoviridae, is anexample of a class of viruses amenable to this approach. These virusescontain 100,000 to 200,000 base pairs of DNA as their genetic material.Importantly, several regions of the genome have been identified that arenonessential for the replication of virus in vitro in cell culture.Modifications in these regions of the DNA may lower the pathogenicity ofthe virus, i.e., attenuate the virus. For example, inactivation of thethymidine kinase gene renders human herpes simplex virus non-pathogenic(28), and pseudorabies virus of swine non-pathogenic (29).

Removal of part of the repeat region renders human herpes simplex virusnon-pathogenic (30,31). A repeat region has been identified in marek'sdisease virus that is associated with viral oncogenicity (32). A regionin herpesvirus saimiri has similarly been correlated with oncogenicity(33). Removal of part of the repeat region renders pseudorabies virusnon-pathogenic (U.S. Pat. No. 4,877,737, issued Oct. 31, 1989). A regionin pseudorabies virus has been shown to be deleted innaturally-occurring vaccine strains (11,3) and it has been shown thatthese deletions are at least partly responsible for the lack ofpathogenicity of these strains.

It is generally agreed that herpesviruses contain non-essential regionsof DNA in various parts of the genome, and that modifications of theseregions can attenuate the virus, leading to a non-pathogenic strain fromwhich a vaccine may be derived. The degree of attenuation of the virusis important to the utility of the virus as a vaccine. Deletions whichcause too much attenuation of the virus will result in a vaccine thatfails to elicit an adequate immune-response. Although several examplesof attenuating deletions are known, the appropriate combination ofdeletions is not readily apparent.

Infectious bovine rhinotracheitis (IBR) virus, an alpha-herpesvirus witha class D genome, is an important pathogen of cattle. It has beenassociated with respiratory, ocular, reproductive, central nervoussystem, enteric, neonatal, and dermal diseases (34). Cattle are thenormal hosts of infectious bovine rhinotracheitis virus, however it alsoinfects goats, swine, water buffalo, wildebeest, mink, and ferrets.Experimental infections have been established in mule deer, goats,swine, ferrets, and rabbits (35).

Conventional modified live virus vaccines have been widely used tocontrol diseases caused by infectious bovine rhinotracheitis virus.However, these vaccine viruses may revert to virulence. More recently,killed virus infectious bovine rhinotracheitis vaccines have been used,but their efficacy appears to be marginal.

Infectious bovine rhinotracheitis virus has been analyzed at themolecular level as reviewed in Ludwig (36). A restriction map of thegenome is available in this reference, which will aid in the geneticengineering of infectious bovine rhinotracheitis according to themethods provided by the present invention.

As reported in the current literature, infectious bovine rhinotracheitisvirus has been engineered to contain a thymidine kinase deletion (43,44)and a deletion in the gIII gene (45,46). However, no evidence has beenpresented for the deletions in the US2, repeat, gG, or gE regions. Inthe subject application, usefulness of such deletions for both theattenuation of infectious bovine rhinotracheitis virus and for thedevelopment of gene deleted marker vaccines is demonstrated.

As with other herpesviruses, infectious bovine rhinotracheitis virus canbecome latent in healthy animals which makes them potential carriers ofthe virus. For this reason it is clearly advantageous to be able todistinguish animals vaccinated with non-virulent virus from animalsinfected with disease-causing wild type virus. The development ofdifferential vaccines and companion diagnostic tests has proven valuablein the management of pseudorabies disease (47). A similar differentialmarker vaccine would be of great value in the management of infectiousbovine rhinotracheitis disease. The construction of differentialdiagnostics has focused on the deletion of glycoproteins. Theoretically,the glycoprotein chosen to be the diagnostic marker should have thefollowing characteristics: (1) the glycoprotein and its gene should benon-essential for the production of infectious virus in tissue culture;(2) the glycoprotein should elicit a major serological response in theanimal; and (3) the glycoprotein should not be one that makes asignificant contribution to the protective immunity. Four majorinfectious bovine rhinotracheitis virus glycoproteins (gI, gII, gIII,and gIV) have been described in the literature (48). Three of thesegenes, gI, gIII, and gIV, have been sequenced and shown to be homologousto the HSV glycoproteins gB, gC, and gD, respectively. Although it hasbeen suggested that the gII protein is analogous to HSV gE, no sequenceevidence has been presented to confirm that suggestion (48). The gB andgD homologues are essential genes and would not be appropriate asdeletion marker genes. The gC gene of herpesviruses has been shown tomake a significant contribution to protective immunity as a target ofneutralizing antibody (49) and as a target of cell-mediated immunity(50). Therefore, the gC gene is not desirable as a deletion marker gene.As indicated above, Kit et al. (45) have described the deletion of theinfectious bovine rhinotracheitis virus gIII as a marker gene. It wouldbe expected that such a deletion would compromise the efficacy of aninfectious bovine rhinotracheitis vaccine.

For pseudorabies virus (PRV) the criteria for a deletion marker gene arebest met by the glycoprotein X (51). Wirth et al. (52) suggests theexistence of a “gX homologue of HSV-1” in the infectious bovinerhinotracheitis virus. It is not clear what is meant by this becausealthough there is a PRV gX gene, there is no reported HSV-1 gX gene orgX homologous gene. In any case, no sequence evidence is presented tosupport this suggestion. Clear evidence of homologues of PRV gX (HSV-2gG) and PRV gI (HSV gE) in infectious bovine rhinotracheitis virus andtheir usefulness as diagnostic markers is demonstrated.

The present invention provides a method of producing a fetal-safe, liverecombinant infectious bovine rhinotracheitis virus which comprisestreating viral DNA from a naturally-occurring live infectious bovinerhinotracheitis virus so as to delete from the virus DNA correspondingto the US2 region of the naturally-occurring infectious bovinerhinotracheitis virus. The present invention also provides viruses inwhich (1) DNA corresponding to the US2 region of naturally-occurringinfectious bovine rhinotracheitis virus has been deleted, and (2) DNAencoding gG and/or gE has been altered or deleted. Such viruses areuseful in vaccines which need diagnostic markers and are safe for use inpregnant animals.

The ability to engineer DNA viruses with large genomes, such as vacciniavirus and the herpesviruses, has led to the finding that theserecombinant viruses can be used as vectors to deliver immunogens toanimals (53). The herpesviruses are attractive candidates fordevelopment as vectors because their host range is primarily limited toa single target species (54), and they have the capacity forestablishing a latent infection (55) that could provide for stable invivo expression of a desired cloned polypeptide. Herpesviruses have beenengineered to express a variety of foreign gene products, such as bovinegrowth hormone (56), human tissue plasminogen activator (57), and E.coli β-galactosidase (58,59). In addition, possible immunogenicpolypeptides have been expressed by herpesviruses. Whealy et al. (60)expressed portions of the human immunodeficiency virus type 1 envelopeglycoprotein in pseudorabies virus (PRV) as fusions to the PRVglycoprotein III. The hepatitis B virus surface antigen (61) and ahybrid human malaria antigen from Plasmodium falciparum have beenexpressed in herpes simplex virus type 1 (HSV-1) (62). The infectiousbovine rhinotracheitis viruses described above may be used as vectorsfor the insertion of genes encoding antigens from microorganisms causingimportant cattle diseases. Such recombinant viruses would be multivalentvaccines protecting against infectious bovine rhinotracheitis as well asother diseases. Kit et al. (63) have described the expression of a Footand Mouth disease antigen in infectious bovine rhinotracheitis virus. Insome of the prior applications from which the subject application claimspriority (which precedes the Kit publication by at least three years),the use of infectious bovine rhinotracheitis virus to express severalforeign genes including the E. coli β-galactosidase (lacZ) gene, the TN5neomycin resistance gene, and antigens from bovine rota virus, andparainfluenza type 3 virus (see U.S. Ser. No. 06/933,107, filed Nov. 20,1986, now abandoned and U.S. Ser. No. 07/078,519, filed Jul. 27, 1987,now abandoned) is described. These applications precede the Kitpublication by at least three years. The viruses described in thisapplication provide a combination of attenuation, differentiation andmultivalency. These properties make such viruses useful as vaccines forthe management of cattle diseases.

SUMMARY OF THE INVENTION

The present invention provides a recombinant infectious bovinerhinotracheitis designated S-IBR-052 (ATCC Accession No. VR 2443). Thepresent invention also provides a vaccine which comprises an effectiveimmunizing amount of the recombinant infectious bovine rhinotracheitisvirus designated S-IBR-052 and a suitable carrier.

The present invention provides homology vectors, methods of immunizationand a method of distinguishing an animal vaccinated with the vaccines ofthe present invention from an animal infected with a naturally-occurringinfectious bovine rhinotracheitis virus.

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1: Details of the infectious bovine rhinotracheitis Cooper Strain.Diagram of infectious bovine rhinotracheitis genomic DNA showing theunique long, internal repeat, unique short, and Terminal repeat regions.Restriction maps for the enzymes HindIII, EcoRI, and XbaI are indicated(7). Fragments are lettered in order of decreasing size. The uniqueshort region is also expanded for inclusion of more detail. The locationof several genes is also indicated, they are unique short 2 (US2),immediate early genes (IE) (20), glycoprotein G (gG), glycoprotein IV(gIV) (17), glycoprotein E (gE). Note that due to the inversion of theshort region, which includes the unique short, internal, and terminalrepeats, four half molar HindIII fragments are present (HindIII D, C, F,and H).

FIG. 2: Details of S-IBR-002. Diagram of S-IBR-002 genomic DNA showingthe unique long, internal repeat, unique short, and Terminal repeatregions. Restriction maps for the enzymes HindIII, EcoRI, and XbaI areindicated (7). Fragments are lettered in order of decreasing size. TheEcoRI B and F fragments are expanded for inclusion of more detail. The˜800 BP repeat deletions are indicated by deltas. Note that due to theinversion of the short region, which includes the unique short,internal, and terminal repeats, four half molar HindIII fragments arepresent (HindIII D, C, F, and H).

FIG. 3 SEQ ID NO. 1: DNA sequence (SEQ ID NO: 19) of the IBR; UniqueShort 2 gene. The sequence of the first 1080 base pairs of the HindIII Kfragment, reading from the HindIII K/HindIII O junction, are shown. Theunique short 2 (US2) gene is transcribed toward the HindIII K/HindIII Ojunction as indicated in FIG. 1. The sequence has been reversed andcomplemented in order to show the translation start and termination ofUS2 gene.

FIGS. 4A-4B SEQ ID NOS: 2-6:

Homology between the infectious bovine rhinotracheitis US2 (SEQ ID NO.2) protein and the US2 proteins of HSV-1, (SEQ ID NO: 3) PRV (SEQ ID NO:4), HSV-2(SEQ ID NO: 5), and marek's disease virus, (SEQ ID NO. 6).(FIG. 4A) Matrix plot of the amino acid sequence of the invectiousbovine rhinotracheitis US2 protein (309) against the amino acid sequenceof the HSV-1 US2 protein (291) (8). (FIG. 4B) Alignment of the conservedregion between infectious bovine rhinotracheitis US2 protein, HSV-1 US2protein, PRV US2 protein (256 amino acids) (21), HSV-2 US2 protein (291)(9), and marek's disease virus US2 protein (270 amino acids)(SEQ ID NOS:20-24)(1).

FIGS. 5A-5B SEQ ID NOS: 7-9:

Details of the Nasalgen deletion. Diagram of infectious bovinerhinotracheitis genomic DNA showing the unique long, internal repeat,unique short, and terminal repeat regions. A restriction map for theenzyme HindIII is indicated. Fragments are lettered in order ofdecreasing size. The unique short region is expanded for inclusion ofmore detail. The location of the deletion in the Nasalgen HindIII Kfragment is shown. Three regions of DNA sequence are also shown (SEQ IDNOS: 25-27). The first line (SEQ ID NO: 7) shows the first 60 base pairsupstream of the HindIII O/HindIII D junction in the infectious bovinerhinotracheitis Cooper strain. The second line (SEQ ID NO: 8) shows thefirst 60 base pairs upstream of the HindIII K/HindIII D junction in theNasalgen strain. The third line (SEQ ID NO: 9) shows 60base pairsflanking the DNA encoding amino acid 59 of the infectious bovinerhinotracheitis US2 gene in the infectious bovine rhinotracheitis Cooperstrain.

FIG. 6: Details of S-IBR-027. Diagram of S-IBR-027 genomic DNA showingthe unique long, internal repeat, unique short, and terminal repeatregions. Restriction maps for the enzymes HindIII, EcoRI, and XbaI areindicated (7). Fragments are lettered in order of decreasing size. Theunique short region is also expanded for inclusion of more detail. Thelocation of several genes is also indicated, they are unique short 2(US2), immediate early genes (IE) (20), glycoprotein G (gG),glycoprotein IV (gIV) (17), glycoprotein E (gE). The unique short regionand repeat region deletions are indicated by deltas. The location of theapproximately 1200 BP deletion of the US2 gene is shown in the expandedregion. Note that due to the inversion of the short region, whichincludes the unique short, internal, and terminal repeats, four halfmolar HindIII fragments are present (HindIII D, C, F, and H).

FIGS. 7A-7H SEQ ID NOS. 10-15:

Detailed description of the DNA insertion in Homology Vector 129-71.5.Diagram showing the orientation of DNA fragments assembled in plasmid129-71.5. The origin of each fragment is indicated in the table. Thesequences (SEQ ID NOS. 10-15) located at each of the junctions betweenfragments is also shown. The restriction sites used to generate eachfragment as well as synthetic linker sequences which were used to jointhe fragments are described for each junction (SEQ ID NOS: 28-33). Thesynthetic linker sequences are underlined by a heavy bar. The locationof several gene coding regions and regulatory elements is also given.The following two conventions are used: numbers in parentheses, ( ),refer to amino acids, and restriction sites in brackets, [ ], indicatethe remnants of sites which were destroyed during construction. Thefollowing abbreviations are used: polyadenylation signal (pA),infectious bovine rhinotracheitis virus (IBR), Herpes simplex virus type1 (HSV-1), thymidine kinase (TK), neomycin resistance (NEO), bacterialtransposon Tn5 (Tn5).

FIG. 8 SEQ ID NO. 16: DNA sequence of the IBR glycoprotein G gene (SEQID NO: 34). The sequence of approximately 1400 base pairs of the HindIIIK fragment, starting approximately 2800 base pairs downstream of theHindIII K/HindIII O junction, are shown. The glycoprotein G (gG) gene istranscribed away from the HindIII K/HindIII O junction as indicated inFIG. 1. The translational start and termination of the gG gene areindicated.

FIGS. 9A-9B SEQ ID NOS: 17-19:

Homology between the IBR gG protein, the gX protein of PRV and the gGprotein of HSV-2. (FIG. 9A) Matrix plot of the amino acid sequence ofthe IBR gG protein (441) against the amino acid sequence of the PRV gXprotein (498) (12). (FIG. 9B) Alignment of the conserved region betweenIBR gG (SEQ ID NO: 17) protein, PRV (SEQ ID NO: 18) protein, and HSV-2(SEQ ID NO: 19) gG protein (699) (9). Note that IUPAC-IUB BiochemicalNomenclature Commission conventions are used.

FIG. 10:

Western blot of proteins released into the medium of IBR and PRVinfected cells, showing the absence of gG in S-PRV-013, S-IBR-035,S-IBR-036, S-IBR-037, and S-IBR-038 but its presence in S-PRV-160 andwild type S-IBR-000. Lanes (A) 0.5 μg purified gG, (B) blank lane, (C)S-PRV-160, (D) S-PRV-013, (E) pre-stained molecular weight markers, (F)0.5 μg purified gG, (G) S-IBR-038, (H) S-IBR-037, (I) S-IBR-036, (J)S-IBR-035, (K) S-IBR-000, (L) uninfected MDBK cells, (M) pre-stainedmolecular weight markers. Media samples were prepared as described inthe PREPARATION OF HERPESVIRUS CELL LYSATES. The concentrated media fromthe infection of one 6 cm dish of infected cells was loaded in eachsample lane except for samples S-PRV-013 and S-PRV-160 for which themedia from two 6 cm dishes were loaded.

FIGS. 11A-11H SEQ ID NOS: 20-25:

Detailed description of the DNA insertion in Plasmid 459-12.6. Diagramshowing the orientation of DNA fragments assembled in plasmid 459-12.6.The origin of each fragment is indicated in the table. The sequences(SEQ ID NOS: 20-25) located at each of the junctions between fragmentsis also shown. The restriction sites used to generate each fragment aswell as synthetic linker sequences which were used to join the fragmentsare described for each junction (SEQ ID NOS: 38-43). The syntheticlinker sequences are underlined by a heavy bar. The location of severalgene coding regions and regulatory elements is also given. The followingtwo conventions are used: numbers in parentheses, ( ), refer to aminoacids, and restriction sites in brackets, [ ], indicate the remnants ofsites which were destroyed during construction. The followingabbreviations are used: unique glycoprotein G (gG), glycoprotein III(gIII), glycoprotein X (gX), polyadenylation signal (pA), infectiousbovine rhinotracheitis virus (IBR), pseudorabies virus (PRV), and humancytomegalovirus (HCMV).

FIGS. 12A-12H SEQ ID NOS. 26, 27 and 29-32:

Detailed description of the DNA insertion in Homology Vector 439-01.31.Diagram showing the orientation of DNA fragments assembled in plasmid439-01.31. The origin of each fragment is indicated in the table. Thesequences (SEQ ID NOS. 26, 27 and 29-32) located at each of thejunctions between fragments is also shown. The restriction sites used togenerate each fragment as well as synthetic linker sequences which wereused to join the fragments are described for each junction (SEQ ID NOS:44-49). The synthetic linker sequences are underlined by a heavy bar.The location of several gene coding regions and regulatory elements isalso given. The following two conventions are used: numbers inparentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which were destroyedduring construction. The following abbreviations are used: unique short2 (US2), glycoprotein G (gG), glycoprotein IV (gIV), polyadenylationsignal (pA), infectious bovine rhinotracheitis virus (IBR), pseudorabiesvirus (PRV), and human cytomegalovirus (HCMV).

FIGS 13A-13H SEQ ID NOS. 29-32, 33 and 34:

Detailed description of the DNA insertion in Homology Vector 439-21.69.Diagram showing the orientation of DNA fragments assembled in plasmid439-21.69. The origin of each fragment its indicated in the table. Thesequences (SEQ ID NOS: 29-32, 33 and 34) located at each of thejunctions between fragments is also shown. The restriction sites used togenerate each fragment as well as synthetic linker sequences which wereused to join the fragments are described for each junction (SEQ ID NOS:50-55). The synthetic linker sequences are underlined by a heavy bar.The location of several gene coding regions and regulatory elements isalso given. The following two conventions are used: numbers inparentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which were destroyedduring construction. The following abbreviations are used: unique short2 (US2), glycoprotein G (gG), glycoprotein IV (gIV), polyadenylationsignal (pA), infectious bovine rhinotracheitis virus (IBR), pseudorabiesvirus (PRV), and human cytomegalovirus (HCMV).

FIGS. 14A-14E SEQ ID NOS: 32, 33 and 35:

Detailed description of the DNA insertion in Homology Vector 439-70.4.Diagram showing the orientation of DNA fragments assembled in plasmid439-70.4. The origin of each fragment is indicated in the table. Thesequences (SEQ ID NOS. 32, 33 and 35) located at each of the junctionsbetween fragments is also shown. The restriction sites used to generateeach fragment as well as synthetic linker sequences which were used tojoin the fragments are described for each junction. The synthetic linkersequences are underlined by a heavy bar. The location of several genecoding regions and regulatory elements is also given. The following twoconventions are used: numbers in parentheses, ( ), refer to amino acids,and restriction sites in brackets, [ ], indicate the remnants of siteswhich were destroyed during construction. The following abbreviationsare used: glycoprotein G (gG), glycoprotein IV (gIV), and infectiousbovine rhinotracheitis virus (IBR).

FIGS. 15A-15B SEQ ID NO. 36:

DNA sequence of the IBR glycoprotein E gene (SEQ ID NOS: 59-67. Thesequence of 2038 base pairs of the IBR unique short region, startingapproximately 1325 base pairs upstream in the HindIII K/HindIII Fjunction in the HindIII K fragment, are shown. The glycoprotein E (gE)gene is transcribed toward the HindIII K/HindIII F junction as indicatedin FIG. 1. The translation start and termination of the gE gene areindicated. Note that IUPAC-IUB Biochemical Nomenclature Commissionconventions are used.

FIGS. 16A-16B SEQ ID NO. 37-40:

Homology between the IBR gE (SEQ ID NO. 40) protein and the gE proteinof HSV-1 (SEQ ID NO. 37), the gI protein of VZV (SEQ ID NO. 39), and thegI protein of PRV. (FIG 16A) Matrix plot of the amino acid sequence ofthe IBR gE protein (617) against the amino acid sequence of the PRV (SEQID NO. 38) gI protein (577) (64). (FIG. 16B) Alignment of the conservedregion between IBR gE protein PRV gI protein, and VZV gI protein (SEQ IDNOS. 61-64)(37).

FIGS. 17A-17E SEQ ID NOS: 41-43:

Detailed description of a plasmid containing the gE gene. Diagramshowing the orientation of DNA fragments to be assembled in thegE-containing plasmid. The origin of each fragment is indicated in thetable. The sequences (SEQ ID NOS. 41-43) located at each of thejunctions between fragments are also shown. The restriction sites usedto generate each fragment are described for each junction (SEQ ID NOS.65-67). The location of several gene coding regions and regulatoryelements is also given. The following two conventions are used: numbersin parentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which were destroyedduring construction. The following abbreviations are used: uniqueglycoprotein E (gE), glycoprotein IV (gIV), and infectious bovinerhinotracheitis virus (IBR).

FIGS. 18A-18G SEQ ID NOS. 44-48:

Detailed description of the DNA insertion in the homology vector536-03.5. Diagram showing the orientation of DNA fragments to beassembled in the homology vector. The origin of each fragment isindicated in the table. The sequences (SEQ ID NOS. 44-48) located ateach of the junctions between fragments is also shown. The restrictionsites used to generate each fragment as well as synthetic linkersequences which were used to join the fragments are described for eachjunction (SEQ ID NOS: 68-72). The synthetic linker sequences areunderlined by a heavy bar. The location of several gene coding regionsand regulatory elements is also given. The following two conventions areused: numbers in parentheses, ( ), refer to amino acids, and restrictionsites in brackets, [ ], indicate the remnants of sites which weredestroyed during construction. The following abbreviations are used:glycoprotein E (gE), immediate early promoter (IE), infectious bovinerhinotracheitis virus (IBR), and pseudorabies virus (PRV).

FIG. 19:

Construction of Recombinant S-IBR-004 Virus. S-IBR-004 is an IBRrecombinant virus carrying an inserted foreign gene (NEO) under thecontrol of the PRV gX promoter. A new XbaI site was created at the shortunique region and the original SacI site was deleted.

FIG. 20:

Construction of Recombinant S-IBR-008 Virus. S-IBR-008 is a recombinantIBR virus that has a bovine rotavirus glycoprotein gene and the plasmidvector inserted in the XbaI site on the unique long region. A sitespecific deletion was created at the [SacI] site due to the loss of NEOgene in the small unique region.

FIGS. 21A-21B SEQ ID NO. 49:

Sequence of the PI-3 (SF-4 Strain) HN Gene (SEQ ID NOS: 73-74). Notethat IUPAC-IUB Biochemical Nomenclature Commission conventions are used.

FIGS. 22A-22C:

Details of S-IBR-018 Construction.

A. First line shows the IBR (Cooper Strain) BamHI-C fragment map. Secondline shows the construction of the alpha-4 promoter on the PI-3 HN geneand its insertion into the HindIII site in BamHI-C. Also shown are thebeta-gal and neomycin (NEO) gene constructions under the control of thegX promoter that were put into the XbaI site and used as selectablemarkers to purify the recombinant virus.

B. The BamHI-C fragment map of S-IBR-018 after insertion of the PI-3 HN,beta-gal, and neomycin genes.

C. The S-IBR-018 genome showing the location of the three insertedforeign genes.

Legend: B=BamHI; H=HindIII; X=XbaI; S=StuI; UL=unique long region;US=unique short region; IR=internal repeat region; TR=terminal repeatregion.

FIGS. 23A-23C:

Details of S-IBR-019 Construction.

A. First line shows the IBR (Cooper Strain) BamHI-C fragment map. Secondline shows the construction of the alpha-4 promoter on the PI-3 F geneand its insertion into the HindIII site in BamHI-C. Also shown are thebeta-gal and neomycin (NEO) gene constructions under the control of thegX promoter that were put into the XbaI site and used as selectablemarkers to purify the recombinant virus.

B. The BamHI-C fragment map of S-IBR-019 after insertion of the PI-3 F,beta gal, and neomycin genes.

C. The S-IBR-019 genome showing the location of the three insertedforeign genes.

FIG. 24:

Detailed description of the DNA insertion in Homology Vector 591-21.20.The diagram shows the orientation of DNA fragments assembled in plasmid591-21.20. The origin of each fragment is described in the MATERIALS ANDMETHODS section. The sequences located at the junctions between eachfragment are shown (SEQ ID NOS: 75-77). The restriction sites used togenerate each fragment as well as synthetic linker sequences which wereused to join the fragments are described for each junction. Thesynthetic linker sequences are underlined by a double bar. The locationof the Tk gene coding region is also given. The following twoconventions are used: numbers in parenthesis ( ) refer to amino acids,and restriction sites in brackets [ ] indicate the remnants of siteswhich were destroyed during construction. The following abbreviation isused, infectious bovine rhinotracheitis virus (IBR).

FIGS. 25A-25B:

Detailed description of the marker gene insertion in Homology Vector591-46.12. The diagram shows the orientation of DNA fragments assembledin the marker gene. The origin of each fragment is described in theMATERIALS AND METHODS section. The sequences located at the junctionsbetween each fragment and at the ends of the marker gene are shown. Therestriction sites used to generate each fragment are indicated at theappropriate junction. The location of the uidA gene coding region isalso given. The following two conventions are used: numbers inparenthesis ( ) refer to amino acids, and restriction sites in brackets[ ] indicate the remnants of sites which were destroyed duringconstruction. The following abbreviations are used, pseudorabies virus(PRV), uronidase A gene (uidA), Escherichia coli (E. coli), herpessimplex virus type 1 (HSV-1), poly adenylation signal (pA), andglycoprotein X (gX).

Legend: B=Ba HI; H=HindIII; X=XbaI; S=StuI; UL=unique long region;US=unique short region; IR=internal repeat region; TR=terminal repeatregion.

FIGS. 26A-26B:

Detailed description of the DNA insertion in the homology vector523-78.72. Diagram showing the orientation of DNA fragments to beassembled in the homology vector. The origin of each fragment isindicated in the table. The sequences located at each of the junctionsbetween fragments is also shown. The restriction sites used to generateeach fragment as well as synthetic linker sequences which were used tojoin then fragments are described for each junction (SEQ ID NO: 78). Thesynthetic linker sequences are underlined by a heavy bar. The locationof several gene coding regions and regulatory elements is also given.The following two conventions are used: numbers in parentheses, ( ),refer to amino acids, and restriction sites in brackets, [ ], indicatethe remnants of sites which were destroyed during construction. Thefollowing abbreviations are used: glycoprotein D (gD), glycoprotein E(gE), infectious bovine rhinotracheitis virus (IBRV).

FIG. 27:

Restriction Enzyme Map of the IBRV BamHI C Fragment. The restriction mapshows non-essential regions within the BamHI C fragment (21.1 kb). TheBamHI C fragment contains part of the unique long region, all of theinternal repeat region and part of the unique short region of IBRV. TheHindIII restriction enzyme site within the 3.9 kb ApaI restrictionfragment within the larger BamHI-KpnI subfragment was used as aninsertion site for foreign genes in S-IBR-018, S-IBR-019, S-IBR-069,S-IBR-071, S-IBR-074, S-IBR-086. The EcoRV restriction enzyme sitewithin the smaller KpnI-BamHI subfragment within the repeat region ofIBRV is used as an insertion site for foreign genes in S-IBR-072. TheXbaI restriction enzyme site within the 3.9 kb ApaI restriction fragmentwithin the larger BamHI-KpnI subfragment was used as an insertion sitefor foreign genes in S-IBR-018 and S-IBR-019.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a recombinant infectious bovinerhinotracheitis (IBR) virus designated S-IBR-052 (ATCC Accession No. VR2443).

S-IBR-052 was deposited on Feb. 4, 1994 pursuant to the Budapest Treatyon the International Deposit of Microorganisms for the Purposes ofPatent Procedure with the Patent Culture Depository of the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,U.S.A. under ATCC Accession No. VR 2443.

The present invention provides a recombinant infectious bovinerhinotracheitis virus, further comprising a foreign DNA sequenceinserted into the IBR virus genomic DNA, wherein the foreign DNAsequence is inserted within a BamHI C fragment of the infectious bovinerhinotracheitis virus genomic DNA and is capable of being expressed in ainfectious bovine rhinotracheitis virus infected host cell.

In one embodiment the foreign DNA is inserted within a larger BamHI-KpnIsubfragment of the BamHI C fragment of the infectious bovinerhinotracheitis virus genomic DNA. In another embodiment the foreign DNAsequence is inserted within a HindIII site located within the largerBamHI-KpnI subfragment of the infectious bovine rhinotracheitis virusgenomic DNA. In another embodiment the foreign DNA sequence is insertedwithin a XbaI site located within the larger BamHI-KpnI subfragment ofthe infectious bovine rhinotracheitis virus genomic DNA.

In one embodiment the foreign DNA is inserted within a smallerKpnI-BamHI subfragment of the BamHI C fragment of the infectious bovinerhinotracheitis virus genomic DNA. In another embodiment the foreign DNAsequence is inserted within a EcoRV site located within smallerKpnI-BamHI subfragment of the infectious bovine rhinotracheitis virusgenomic DNA.

In another embodiment the infectious bovine rhinotracheitis virusgenomic DNA contains a deletion or deletions in a non-essential regionof the infectious bovine rhinotracheitis virus. In one embodiment thedeletion is in either the US2, gG or gE gene regions.

In another embodiment the infectious bovine rhinotracheitis virusfurther comprises a foreign DNA sequence inserted into an open readingframe encoding infectious bovine rhinotracheitis virus thymidine kinase.

For purposes of this invention, “a recombinant infectious bovinerhinotracheitis virus capable of replication” is a live infectiousbovine rhinotracheitis virus which has been generated by the recombinantmethods well known to those of skill in the art, e.g., the methods setforth in HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANTHERPESVIRUS in Materials and Methods and has not had genetic materialessential for the replication of the recombinant infectious bovinerhinotracheitis virus deleted.

For purposes of this invention, “an insertion site which is notessential for replication of the infectious bovine rhinotracheitisvirus” is a location in the infectious bovine rhinotracheitis viralgenome where a sequence of DNA is not necessary for viral replication,for example, complex protein binding sequences, sequences which code forreverse transcriptase or an essential glycoprotein, DNA sequencesnecessary for packaging, etc.

For purposes of this invention, an “open reading frame” is a segment ofDNA which contains codons that can be transcribed into RNA which can betranslated into an amino acid sequence and which does not contain atermination codon.

The present invention provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which DNA encoding gGglycoprotein has been altered or deleted so that upon replication therecombinant infectious bovine rhinotracheitis virus produces no gGglycoprotein. The DNA encoding gG glycoprotein may be deleted or foreignDNA may be inserted into the DNA encoding gG glycoprotein. The DNAencoding gG glycoprotein may be deleted and foreign DNA may be insertedin place of the deleted DNA encoding gG glycoprotein.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which DNA encoding gGglycoprotein has been altered or deleted and DNA encoding the gEglycoprotein has been altered or deleted so that upon replication therecombinant infectious bovine rhinotracheitis virus produces no gGglycoprotein and no gE glycoprotein. The DNA encoding gG glycoproteinmay be deleted or foreign DNA may be inserted into the DNA encoding gGglycoprotein. The DNA encoding gE glycoproteian may be deleted andforeign DNA may be inserted in place of the deleted DNA encoding gEglycoprotein.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which DNA encoding gGglycoprotein has been altered or deleted so that upon replication therecombinant infectious bovine rhinotracheitis virus produces no gGglycoprotein, DNA corresponding to the US2 region of thenaturally-occurring infectious bovine rhinotracheitis virus has beendeleted, and DNA encoding the gE glycoprotein has been altered ordeleted.

The present invention also provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which (1) DNA correspondingto the US2 region of the naturally-occurring infectious bovinerhinotracheitis virus has been deleted, and (2) DNA encoding gGglycoprotein has been altered or deleted. The DNA encoding the gGglycoprotein may be deleted or foreign DNA may be inserted in place ofthe deleted DNA encoding gG glycoprotein. Foreign DNA may be inserted inplace of the deleted DNA corresponding to the US2 region of thenaturally-occurring infectious bovine rhinotracheitis virus.

The present invention also provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which (1) DNA correspondingto the US2 region of the naturally-occurring infectious bovinerhinotracheitis virus has been deleted, and (2) DNA encoding gEglycoprotein has been altered or deleted. The DNA encoding the gGglycoprotein may be deleted or foreign DNA may be inserted in place ofthe deleted DNA encoding gE glycoprotein. Foreign DNA may be inserted inplace of the deleted DNA corresponding to the US2 region of thenaturally-occurring infectious bovine rhinotracheitis virus.

The present invention also provides S-IBR-037, a recombinant infectiousbovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus in which (1)DNA corresponding to the US2 region of the naturally-occurringinfectious bovine rhinotracheitis virus has been deleted, and (2) DNAencoding gG glycoprotein has been deleted. S-IBR-037 was deposited onApr. 16, 1991 pursuant to the Budapest Treaty on the InternationalDeposit of Microorganisms for the Purposes of Patent Procedure with thePatent Culture Depository of the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209, U.S.A. under ATCC AccessionNo. VR 2320.

The present invention also provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which (1) DNA correspondingto the US2 region of the naturally-occurring infectious bovinerhinotracheitis virus has been deleted and a foreign DNA sequence whichencodes Escherichia coli β-galactosidase has been inserted in place ofthe deleted DNA encoding gG glycoprotein, and (2) DNA encoding gGglycoprotein has been altered or deleted. The present invention alsoprovides two examples of such viruses, S-IBR-035 and S-IBR-036.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which DNA encoding gEglycoprotein has been altered or deleted so that upon replication therecombinant infectious bovine rhinotracheitis virus produces no gEglycoprotein. The DNA encoding gE glycoprotein may be deleted or foreignDNA may be inserted in the DNA encoding gE glycoprotein. The DNAencoding gE glycoprotein may be deleted and foreign DNA may be insertedin place of the deleted DNA encoding gE glycoprotein.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which DNA encoding gEglycoprotein has been altered or deleted so that upon replication therecombinant infectious bovine rhinotracheitis virus produces no gEglycoprotein and DNA corresponding to the US2 region of thenaturally-occurring infectious bovine rhinotracheitis virus has beendeleted.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus from which DNA in the uniqueshort region of the naturally-occurring infectious bovinerhinotracheitis virus has been deleted. Foreign DNA may be inserted intothe DNA of the recombinant infectious bovine rhinotracheitis virus. Theforeign DNA may be inserted into the XbaI site in the long uniqueregion. The foreign DNA may be a sequence which encodes bovine rotavirusglycoprotein 38; this sequence may be inserted into the XbaI site in thelong unique region.

The present invention provides S-IBR-008, a recombinant infectiousbovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus from whichDNA corresponding to the unique short region of the naturally-occurringinfectious bovine rhinotracheitis virus has been deleted and in which aforeign DNA sequence which encodes bovine rotavirus glycoprotein 38 hasbeen inserted into the XbaI site in the long unique region. S-IBR-008was deposited on Jun. 18, 1986 pursuant to the Budapest Treaty on theInternational Deposit of Microorganisms for the Purposes of PatentProcedure with the Patent Culture Depository of the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,U.S.A. under ATCC Accession No. VR 2141.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus from which (1) DNA correspondingto the US2 region of the naturally-occurring infectious bovinerhinotracheitis virus has been deleted and (2) at least a portion ofboth repeat sequences has been deleted. The present invention furtherprovides an example of such a recombinant virus, designated S-IBR-027.S-IBR-027 was deposited on Apr. 17, 1991 pursuant to the Budapest Treatyon the International Deposit of Microorganisms for the Purposes ofPatent Procedure with the Patent Culture Depository of the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,U.S.A. under ATCC Accession No. VR 2322.

The present invention further provides a recombinant IBR viruscomprising viral DNA from a naturally-occurring IBR virus from which atleast a portion of both repeat sequences has been deleted.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringIBR virus from which (1) at least a portion of both repeat sequences hasbeen deleted and (2) DNA encoding one or more EcoRV restriction siteshas been deleted. The present invention further provides an example ofsuch a recombinant virus, designated S-IBR-002. S-IBR-002 was depositedon Jun. 18, 1986 pursuant to the Budapest Treaty on the InternationalDeposit of Microorganisms for the Purposes of Patent Procedure with thePatent Culture Depository of the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209, U.S.A. under ATCC AccessionNo. VR 2140.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus comprising. viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus from which (1) at least aportion of both repeat sequences has been deleted and (2) whereinforeign DNA has been inserted into the DNA of the recombinant infectiousbovine rhinotracheitis virus. The foreign DNA may be a sequence whichencodes the Tn5 NEO gene.

The present invention further provides S-IBR-020, a recombinantinfectious bovine rhinotracheitis virus comprising viral DNA from anaturally-occurring IBR virus from which (1) at least a portion of bothrepeat sequences has been deleted and (2) wherein a foreign DNA sequencewhich encodes the Tn5 NEO gene has been inserted into the DNA of therecombinant infectious bovine rhinotracheitis virus.

The present invention also provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus from which (1) at least aportion of both repeat sequences has been deleted, (2) wherein a foreignDNA sequence which encodes the Tn5 NEO gene has been inserted into theDNA of the recombinant infectious bovine rhinotracheitis virus, and (3)wherein at least a portion of the thymidine kinase gene has beendeleted.

The present invention also provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus from which (1) at least aportion of both repeat sequences has been deleted, (2) wherein a foreignDNA sequence which encodes the Tn5 NEO gene has been inserted into theDNA of the recombinant infectious bovine rhinotracheitis virus, and (3)wherein at least a portion of the thymidine kinase gene has beendeleted. The subject invention provides an example of such a recombinantvirus, designated S-IBR-028. S-IBR-028 was deposited on May 14, 1991pursuant to the Budapest Treaty on the International Deposit ofMicroorganisms for the Purposes of Patent Procedure with the PatentCulture Depository of the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209, U.S.A. under ATCC AccessionNo. VR 2326.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringIBR virus in which a foreign DNA sequence which encodes the Tn5 NEO genehas been inserted into the viral DNA. The Tn5 NEO gene may be under thecontrol of an inserted, upstream, pseudorabies virus glycoprotein Xpromoter. The subject invention further provides an example of arecombinant virus wherein the Tn5 NEO gene is under the control of aninserted, upstream, pseudorabies virus glycoprotein X promoter,designated S-IBR-004. S-IBR-004 was deposited on May 23, 1986 pursuantto the Budapest Treaty on the International Deposit of Microorganismsfor the Purposes of Patent Procedure with the Patent Culture Depositoryof the American Type Culture Collection, 10801 University Blvd.,Manassas, Va. 20110-2209, U.S.A. under ATCC Accession No. VR 2134.

The subject invention further provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which a foreign DNA sequencewhich encodes the Escherichia coli β-galactosidase and Tn5 NEO genes,and the parainfluenza type 3 virus hemagglutinin gene, HN, has beeninserted into the viral DNA. The subject invention provides an exampleof such a recombinant virus, designated S-IBR-018.

The subject invention further provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which a foreign DNA sequencewhich encodes the Escherichia coli β-galactosidase and Tn5 NEO genes,and the parainfluenza type 3 virus fusion gene, F, has been insertedinto the viral DNA. The subject invention provides an example of such arecombinant virus, designated S-IBR-019.

The recombinant viruses of the subject invention were derived from theCooper Strain. However, other infectious bovine rhinotracheitis viruses,such as the LA strain or the 3156 strain, may also be used.

The invention further provides a foreign DNA sequence or foreign RNAwhich encodes a polypeptide. Preferably, the polypeptide is antigenic inthe animal. Preferably, this antigenic polypeptide is a linear polymerof more than 10 amino acids linked by peptide bonds which stimulates theanimal to produce antibodies.

The invention further provides a recombinant infectious bovinerhinotracheitis virus capable of replication which contains a foreignDNA encoding a polypeptide which is a detectable marker. Preferably thedetectable marker is the polypeptide E. coli β-galactosidase.

For purposes of this invention, a “polypeptide which is a detectablemarker” includes the bimer, trimer and tetramer form of the polypeptide.E. coli β-galactosidase is a tetramer composed of four polypeptides ormonomer sub-units.

The invention further provides a recombinant infectious bovinerhinotracheitis virus capable of replication which contains foreign DNAencoding an antigenic polypeptide which is or is from Serpulinahyodysenteriae, Foot and Mouth Disease Virus, Hog Cholera Virus, SwineInfluenza Virus, African Swine Fever Virus or Mycoplasma hyopneumoniae.

The invention further provides for a recombinant infectious bovinerhinotracheitis virus capable of replication which contains foreign DNAencoding pseudorabies virus (PRV) g50 (gD). This recombinant infectiousbovine rhinotracheitis virus can be further engineered to containforeign DNA encoding a detectable marker, such as E. coliβ-galactosidase.

In one embodiment of the recombinant infectious bovine rhinotracheitisvirus the foreign DNA sequence encodes a cytokine. In another embodimentthe cytokine is chicken myelomonocytic growth factor (cMGF) or chickeninterferon (cIFN). Cytokines include, but are not limited to:transforming growth factor beta, epidermal growth factor family,fibroblast growth factors, hepatocyte growth factor, insulin-like growthfactors, B-nerve growth factor, platelet-derived growth factor, vascularendothelial growth factor, interleukin 1, IL-1 receptor antagonist,interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin6, IL-6 soluble receptor, interleukin 7, interleukin 8, interleukin 9,interleukin 10, interleukin 11, interleukin 12, interleukin 13,angiogenin, chemokines, colony stimulating factors,granulocyte-macrophage colony stimulating factors, erythropoietin,interferon, interferon gamma, leukemia inhibitory factor, oncostatin M,pleiotrophin, secretory leukocyte protease inhibitor, stem cell factor,tumor necrosis factors, and soluble TNF receptors. These cytokines arefrom humans, bovine, equine, feline, canine, porcine or avian.Recombinant IBRV expressing cytokines is useful to enhance the immuneresponse when combined with vaccines containing anitgens of diseasecausing microorganisms.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus which comprises a foreign DNA sequence insertedinto a non-essential site of the infectious bovine rhinotracheitis virusgenome, wherein the foreign DNA sequence encodes an antigenicpolypeptide derived from a human pathogen and is capable of beingexpressed in a host infected by the recombinant infectious bovinerhinotracheitis virus.

Recombinant infectious bovine rhinotracheitis virus expressing cytokinesis used to enhance the immune response either alone or when combinedwith vaccines containing cytokines or antigen genes of disease causingmicroorganisms.

Antigenic polypeptide of a human pathogen which are derived from humanherpesvirus include, but are not limited to: hepatitis B virus andhepatitis C virus hepatitis B virus surface and core antigens, hepatitisC virus, human immunodeficiency virus, herpes simplex virus-1, herpessimplex virus-2, human cytomegalovirus, Epstein-Barr virus,Varicella-Zoster virus, human herpesvirus-6, human herpesvirus-7, humaninfluenza, measles virus, hantaan virus, pneumonia virus, rhinovirus,poliovirus, human respiratory syncytial virus, retrovirus, human T-cellleukemia virus, rabies virus, mumps virus, malaria (Plasmodiumfalciparum), Bordetella pertussis, Diptheria, Rickettsia prowazekii,Borrelia berfdorferi, Tetanus toxoid, malignant tumor antigens.

In one embodiment of the invention, a recombinant infectious bovinerhinotracheitis virus contains the foreign DNA sequence encodinghepatitis B virus core protein.

The antigenic polypeptide of an equine pathogen is derived from equineinfluenza virus, or equine herpesvirus. In one embodiment the antigenicpolypeptide is equine influenza neuraminidase or hemagglutinin. Examplesof such antigenic polypeptide are: equine influenza virus type A/Alaska91 neuraminidase and hemagglutinin, equine influenza virus type A/Prague56 neuraminidase and hemagglutinin, equine influenza virus type A/Miami63 neuraminidase, equine influenza virus type A/Kentucky 81neuraminidase and hemagglutinin, equine herpesvirus type 1 glycoproteinB, and equine herpesvirus type 1 glycoprotein D, Streptococcus equi,equine infectious anemia virus, equine encephalitis virus, equinerhinovirus and equine rotavirus.

The present invention further provides an antigenic polypeptide whichincludes, but is not limited to: hog cholera virus gE1, hog choleravirus gE2, swine influenza virus hemagglutinin, neuraminidase, matrixand nucleoprotein, pseudorabies virus gB, gC and gD, and PRRS virusORF7.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus which comprises a foreign DNA sequence insertedinto a non-essential site of the infectious bovine rhinotracheitis virusgenome, wherein the foreign DNA sequence encodes an antigenicpolypeptide derived from bovine respiratory syncytial virus or bovineparainfluenza virus, and is capable of being expressed in a hostinfected by the recombinant infectious bovine rhinotracheitis virus.

For example, the antigenic polypeptide is derived from bovinerespiratory syncytial virus attachment protein (BRSV G), bovinerespiratory syncytial virus fusion protein (BRSV F), bovine respiratorysyncytial virus nucleocapsid protein (BRSV N), bovine parainfluenzavirus type 3 fusion protein, and the bovine parainfluenza virus type 3hemagglutinin neuraminidase.

In one embodiment the recombinant infectious bovine rhinotracheitisvirus is designated S-IBR-071. In another embodiment the recombinantinfectious bovine rhinotracheitis virus is designated S-IBR-072. Inanother embodiment the recombinant infectious bovine rhinotracheitisvirus is designated S-IBR-073.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus which comprises a foreign DNA sequence insertedinto a non-essential site of the infectious bovine rhinotracheitisgenome, wherein the foreign DNA sequence encodes bovine viral diarrheavirus (BVDV) glycoprotein 48 or glycoprotein 53, and wherein the foreignDNA sequence is capable of being expressed in a host infected by therecombinant infectious bovine rhinotracheitis virus.

In one embodiment the recombinant infectious bovine rhinotracheitisvirus is designated S-IBR-069. In another embodiment the recombinantinfectious bovine rhinotracheitis virus is designated S-IBR-074. Inanother embodiment the recombinant infectious bovine rhinotracheitisvirus is designated S-IBR-086.

The present invention further provides a recombinant infectious bovinerhinotracheitis virus in which the foreign DNA sequence encodes anantigenic polypeptide which includes, but is not limited to: Marek'sdisease virus (MDV) gA, marek's disease virus gB, Marek's disease virusgD, Newcastle disease virus (NDV) HN, Newcastle disease virus F,infectious laryngotracheitis virus (ILT) gB, infectiouslaryngotracheitis virus gI, infectious laryngotracheitis virus gD,infectious bursal disease virus (IBDV) VP2, infectious bursal diseasevirus VP3, infectious bursal disease virus VP4, infectious bursaldisease virus polyprotein, infectious bronchitis virus (IBV) spike,infectious bronchitis virus matrix, avian encephalomyelitis virus, avianreovirus, avian paramyxovirus, avian influenza virus, avian adenovirus,fowl pox virus, avian coronavirus, avian rotavirus, chick anemia virus,Salmonella spp. E. coli, Pasteurella spp., Bordetella spp., Eimeriaspp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes,trematodes, poultry mites/lice, and poultry protozoa.

The invention further provides that the inserted foreign DNA sequence isunder the control of an endogenous infectious bovine rhinotracheitisvirus promoter. In one embodiment the foreign DNA sequence is undercontrol of a heterologous promoter. In another embodiment theheterologous promoter is a herepesvirus promoter.

For purposes of this invention, promoters include but is not limited to:herpes simplex virus type I ICP4 protein promoter, a pseudorabies virusglycoprotein X promoter, PRV gX, HCMV immediate early, marek's diseasevirus gA, marek's disease virus gB, marek's disease virus gD, infectiouslaryngotracheitis virus gB, BHV-1.1 VP8 and infectious laryngotracheitisvirus gD.

The subject invention also provides a vaccine which comprises a suitablecarrier and an effective immunizing amount of any of the recombinantviruses of the present invention. The vaccine may contain eitherinactivated or live recombinant virus.

Suitable carriers for the recombinant virus are well known in the artand include proteins, sugars, etc. One example of such a suitablecarrier is a physiologically balanced culture medium containing one ormore stabilizing agents such as hydrolyzed proteins, lactose, etc.Preferably, the live vaccine is created by taking tissue culture fluidsand adding stabilizing agents such as stabilized, hydrolyzed proteins.Preferably, the inactivated vaccine uses tissue culture fluids directlyafter inactivation of the virus.

The subject invention also provides a vaccine which comprises a suitablecarrier and an effective immunizing amount of a recombinant viruscomprising viral DNA from a naturally-occurring infectious bovinerhinotracheitis virus in which DNA encoding gG glycoprotein has-beenaltered or deleted so that upon replication the recombinant infectiousbovine rhinotracheitis virus produces no gG glycoprotein.

The subject invention provides a vaccine which comprises a suitablecarrier and an effective immunizing amount of a recombinant infectiousbovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus in which DNAencoding gG glycoprotein has been altered or deleted and DNA encodingthe gE glycoprotein has been altered or deleted so that upon replicationthe recombinant infectious bovine rhinotracheitis virus produces no gGglycoprotein and no gE glycoprotein.

The subject invention also provides a vaccine which comprises a suitablecarrier and an effective immunizing amount of a recombinant infectiousbovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus in which DNAencoding gG glycoprotein has been altered or deleted so that uponreplication the recombinant infectious bovine rhinotracheitis virusproduces no gG glycoprotein, DNA corresponding to the US2 region of thenaturally-occurring infectious bovine rhinotracheitis virus has beendeleted, and DNA encoding the gE glycoprotein has been altered ordeleted.

The subject invention further provides a vaccine which comprises asuitable carrier and an effective immunizing amount of a recombinantinfectious bovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus in which (1)DNA corresponding to the US2 region of the naturally-occurringinfectious bovine rhinotracheitis virus has been deleted, and (2) DNAencoding gG glycoprotein has been altered or deleted.

The subject invention also provides a vaccine which comprises a suitablecarrier and an effective immunizing amount of a recombinant infectiousbovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus in which DNAencoding gE glycoprotein has been altered or deleted so that uponreplication the recombinant infectious bovine rhinotracheitis virusproduces no gE glycoprotein.

The subject invention provides a vaccine which comprises a suitablecarrier and an effective immunizing amount of a recombinant infectiousbovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus in which DNAencoding gE glycoprotein has been altered or deleted so that uponreplication the recombinant infectious bovine rhinotracheitis virusproduces no gE glycoprotein and DNA corresponding to the US2 region ofthe naturally-occurring infections bovine rhinotracheitis virus has beendeleted.

The subject invention also provides a vaccine which comprises a suitablecarrier and an effective immunizing amount of a recombinant infectiousbovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus from whichDNA corresponding to the US2 region of the naturally-occurringinfectious bovine rhinotracheitis virus has been deleted.

The subject invention provides a vaccine which comprises a suitablecarrier and an effective immunizing amount of a recombinant infectiousbovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus from whichat least a portion of both repeat sequences has been deleted.

The subject invention further provides a vaccine which comprises asuitable carrier and an effective immunizing amount of a recombinantinfectious bovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus in which aforeign DNA sequence which encodes the Tn5 NEO gene has been insertedinto the viral DNA.

The subject invention also provides a vaccine which comprises a suitablecarrier and an effective immunizing amount of a recombinant infectiousbovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus in which aforeign DNA sequence which encodes the Escherichia coli β-galactosidaseand Tn5 NEO genes, and the parainfluenza type 3 virus hemagglutiningene, HN, has been inserted into the viral DNA.

The subject invention also provides a vaccine which comprises a suitablecarrier and an effective immunizing amount of a recombinant infectiousbovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus in which aforeign DNA sequence which encodes the Escherichia coli β-galactosidaseand Tn5 NEO genes, and the parainfluenza type 3 virus fusion gene, F,has been inserted into the viral DNA.

All of the vaccines described hereinabove and hereinbelow may containeither inactivated or live recombinant virus. The vaccines may beadministered by any of the methods well known to those skilled in theart, for example, by intramuscular, subcutaneous, intraperitoneal, orintravenous injection.

Alternatively, the vaccine may be administered intranasally or orally.

The present invention also provides a method of immunizing an animalagainst infectious bovine rhinotracheitis virus which comprisesadministering to the animal an effective immunizing dose of any of thevaccines of the present invention. The animal may be a bovine.

As defined herein an animal includes, but is not limited to: a human,swine, bovine, equine, caprine or ovine. For purposes of this invention,this includes immunizing the animal against the virus or viruses whichcause the disease or diseases: pseudorabies, transmissiblegastroenteritis, swine rotavirus, swine parvovirus, Serpulinahyodysenteriae, bovine viral diarrhea, Newcastle disease, swineinfluenza, PRRS, bovine respiratory synctial virus, bovine parainfluenzavirus type 3, foot and mouth disease, hog cholera, African swine feveror Mycoplasma hyopneumoniae. For purposes of this invention, the methodof immunizing also includes immunizing the animal against humanpathogens, feline pathogens, bovine pathogens, equine pathogens, avianpathogens described in the preceding part of this section.

Recombinant infectious bovine rhinotracheitisV is useful as a vaccineagainst feline or canine diseases when foreign antigens from thefollowing diseases or disease organisms are expressed in the IBRVvector, including but not limited to feline herpesvirus, feline leukemiavirus, feline immunodeficiency virus (FIV) and Dirofilaria immiitis(heartworm). Disease causing microorganisms in dogs include, but are notlimited to canine herpesvirus, canine distemper, canine adenovirus type1 (hepatitis), adenovirus type 2 (respiratory disease), parainfluenza,leptospira canicola, icterohemorragia, parvovirus, coronavirus, Borreliaburgdorferi, canine herpesvirus, Bordetella bronchiseptica, Dirofilariaimmitis (heartworm) and rabies virus. FIV env and gag genes expressed ininfectious bovine rhinotracheitisV is useful as a vaccine against felineimmunodeficiency and is useful to express the FIV env and gag proteinantigens for use in a diagnostic assay. D. immitis p39 and 22kD genesexpressed in IBRV is useful as a vaccine against heartworm in dogs andcats and is useful to express the D. immitis p39 and 22kD proteinantigens for use in a diagnostic assay.

Recombinant IBRV is useful as a vaccine against avian diseases whenforeign antigens from the following diseases or disease organisms areexpressed in the IBRV vector: Chick anemia virus, Avianencephalomyelitis virus, Avian reovirus, Avian paramyxoviruses, Avianinfluenza virus , Avian adenovirus, Fowl pox virus, Avian coronavirus,Avian rotavirus, Salmonella spp E coli, Pasteurella spp, Haemophilusspp, Chlamydia spp, Mycoplasma spp, Campylobacter spp, Bordetella spp,Poultry nematodes, cestodes, trematodes, Poultry mites/lice, Poultryprotozoa (Eimeria spp, Histomonas spp, Trichomonas spp).

Recombinant IBRV is useful as a vaccine against equine diseases whenforeign antigens from the following diseases or disease organisms areexpressed in the IBRV vector: Streptococcus equi, equine infectiousanemia virus, equine encephalitis virus, equine rhinovirus and equinerotavirus.

The method comprises administering to the animal an effective immunizingdose of the vaccine of the present invention. The vaccine may beadministered by any of the methods well known to those skilled in theart, for example, by intramuscular, subcutaneous, intraperitoneal orintravenous injection. Alternatively, the vaccine may be administeredintranasally or orally.

The present invention also provides a host cell infected with arecombinant infectious bovine rhinotracheitis virus capable ofreplication. In one embodiment, the host cell is a mammalian cell.Preferably, the mammalian cell is a Vero cell. Preferably, the mammaliancell is an ESK-4 cell, PK-15 cell or EMSK cell.

For purposes of this invention a “host cell” is a cell used to propagatea vector and its insert. Infecting the cells was accomplished by methodswell known to those of skill in the art, for example, as set forth inINFECTION—TRANSFECTION PROCEDURE in Material and Methods.

Methods for constructing, selecting and purifying recombinant infectiousbovine rhinotracheitis viruses described above are detailed below inMaterials and Methods.

The subject invention also provides a method for distinguishing ananimal vaccinated with a vaccine which comprises an effective immunizingamount of a recombinant virus of the present invention from an animalinfected with a naturally-occurring IBR virus which comprises analyzinga sample of a body fluid from the animal for the presence of gGglycoprotein of infectious bovine rhinotracheitis virus and at least oneother antigen normally expressed in an animal infected by anaturally-occurring infectious bovine rhinotracheitis virus, identifyingantigens which are present in the body fluid, and determining whether gGglycoprotein is present in the body fluid. The presence of antigenswhich are normally expressed in an animal by a naturally-occurringinfectious bovine rhinotracheitis virus and the absence of gGglycoprotein in the body fluid is indicative of an animal vaccinatedwith the vaccine and not infected with a naturally-occurring infectiousbovine rhinotracheitis virus. The presence of antigens and gGglycoprotein in the body fluid may be determined by detecting in thebody fluid antibodies specific for the antigens and gG glycoprotein.

One of the vaccines that is useful in this method is a vaccine whichcomprises a suitable carrier and an effective immunizing amount of arecombinant virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which DNA encoding gGglycoprotein has been altered or deleted so that upon replication therecombinant infectious bovine rhinotracheitis virus produces no gGglycoprotein. Another vaccine that is useful in this method is a vaccinewhich comprises a suitable carrier and an effective immunizing amount ofa recombinant infectious bovine rhinotracheitis virus comprising viralDNA from a naturally-occurring infectious bovine rhinotracheitis virusin which DNA encoding gG glycoprotein has been altered or deleted andDNA encoding the gE glycoprotein has been altered or deleted so thatupon replication the recombinant infectious bovine rhinotracheitis virusproduces no gG glycoprotein and no gE glycoprotein. Yet another vaccinethat is useful in this method is a vaccine which comprises a suitablecarrier and an effective immunizing amount of a recombinant infectiousbovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus in which DNAencoding gG glycoprotein has been altered or deleted so that uponreplication the recombinant infectious bovine rhinotracheitis virusproduces no gG glycoprotein, DNA corresponding to the US2 region of thenaturally-occurring infectious bovine rhinotracheitis virus has beendeleted, and DNA encoding the gE glycoprotein has been altered ordeleted. Still another vaccine that is useful in this method is avaccine which comprises a suitable carrier and an effective immunizingamount of a recombinant infectious bovine rhinotracheitis viruscomprising viral DNA from a naturally-occurring infectious bovinerhinotracheitis virus in which (1) DNA corresponding to the US2 regionof the naturally-occurring infectious bovine rhinotracheitis virus hasbeen deleted, and (2) DNA encoding gG glycoprotein has been altered ordeleted.

The present invention also provides a method for distinguishing ananimal vaccinated with a vaccine which comprises an effective immunizingamount of a recombinant virus of the present invention from an animalinfected with a naturally-occurring infectious bovine rhinotracheitisvirus which comprises analyzing a sample of a body fluid from the animalfor the presence of gE glycoprotein of infectious bovine rhinotracheitisvirus and at least one other antigen normally expressed in an animalinfected by a naturally-occurring infectious bovine rhinotracheitisvirus, identifying antigens which are present in the body fluid anddetermining whether gE glycoprotein is present in the body fluid. Thepresence of antigens which are normally expressed in an animal by anaturally-occurring infectious bovine rhinotracheitis. virus and theabsence of gE glycoprotein in the body fluid is indicative of an animalvaccinated with the vaccine and not infected with a naturally-occurringinfectious bovine rhinotracheitis virus. The presence of antigens and gEglycoprotein in the body fluid may be determined by detecting in thebody fluid antibodies specific for the antigens and gE glycoprotein.

One of the vaccines useful in this method is a vaccine which comprises asuitable carrier and an effective immunizing amount of a recombinantinfectious bovine rhinotracheitis virus comprising viral DNA from anaturally-occurring infectious bovine rhinotracheitis virus in which DNAencoding gG glycoprotein has been altered or deleted and DNA encodingthe gE glycoprotein has been altered or deleted so that upon replicationthe recombinant infectious bovine rhinotracheitis virus produces no gGglycoprotein and no gE glycoprotein. Another vaccine that is useful inthis method is a vaccine which comprises a suitable carrier and aneffective immunizing amount of a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which DNA encoding gGglycoprotein has been altered or deleted so that upon replication therecombinant infectious bovine rhinotracheitis virus produces no gGglycoprotein, DNA corresponding to the US2 region of thenaturally-occurring infectious bovine rhinotracheitis virus has beendeleted, and DNA encoding the gE glycoprotein has been altered ordeleted. Yet another vaccine that is useful in this method is a vaccinewhich comprises a suitable carrier and an effective immunizing amount ofa recombinant infectious bovine rhinotracheitis virus comprising viralDNA from a naturally-occurring infectious bovine rhinotracheitis virusin which DNA encoding gE glycoprotein has been altered or deleted sothat upon replication the recombinant infectious bovine rhinotracheitisvirus produces no gE glycoprotein. Still another vaccine that is usefulin this method is a vaccine which comprises a suitable carrier and aneffective immunizing amount of a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus in which DNA encoding gEglycoprotein has been altered or deleted so that upon replication therecombinant infectious bovine rhinotracheitis virus produces no gEglycoprotein and DNA corresponding to the US2 region of thenaturally-occurring infectious bovine rhinotracheitis virus has beendeleted.

The present invention also provides isolated DNA encoding the gGglycoprotein of infectious bovine rhinotracheitis virus. The subjectinvention also provides purified recombinant gG glycoprotein encoded bythe DNA encoding the gG glycoprotein of infectious bovinerhinotracheitis virus. The subject invention further provides arecombinant cloning vector which comprises the DNA encoding the gGglycoprotein of infectious bovine rhinotracheitis virus. The subjectinvention also provides a recombinant expression vector which comprisesthe DNA encoding the gG glycoprotein of infectious bovinerhinotracheitis virus. The subject invention provides a host cell whichcomprises the recombinant expression vector which comprises the DNAencoding the gG glycoprotein of infectious bovine rhinotracheitis virus.

The subject invention also provides a method of producing a polypeptidewhich comprises growing the host cell which comprises the recombinantexpression vector which comprises the DNA encoding the gG glycoproteinof infectious bovine rhinotracheitis virus under conditions such thatthe recombinant expression vector expresses gG glycoprotein andrecovering the gG glycoprotein so expressed.

The subject invention also provides an antibody directed to an epitopeof the purified gG glycoprotein of infectious bovine rhinotracheitisvirus encoded by the DNA encoding the gG glycoprotein of infectiousbovine rhinotracheitis virus. The antibody may be a monoclonal antibody.

The subject invention also provides a method of detecting the presenceor absence of gG glycoprotein of infectious bovine rhinotracheitis virusin a sample which comprises contacting the sample with an antibodydirected to an epitope of the purified gG glycoprotein of infectiousbovine rhinotracheitis virus encoded by the DNA encoding the gGglycoprotein of infectious bovine rhinotracheitis virus under conditionssuch that the antibody forms a complex with any gG glycoprotein presentin the sample and detecting the presence or absence of such complex. Thesample may be bovine-derived.

The subject invention also provides isolated DNA encoding the gEglycoprotein of infectious bovine rhinotracheitis virus. The subjectinvention also provides purified recombinant gE glycoprotein encoded bythe DNA encoding the gE glycoprotein of infectious bovinerhinotracheitis virus. The subject invention further provides arecombinant cloning vector which comprises the DNA encoding the gEglycoprotein of infectious bovine rhinotracheitis virus. The subjectinvention provides a recombinant expression vector which comprises theDNA encoding the gE glycoprotein of infectious bovine rhinotracheitisvirus. The subject invention also provides a host cell which comprisesthe recombinant expression vector which comprises the DNA encoding thegE glycoprotein of infectious bovine rhinotracheitis virus.

The subject invention also provides a method of producing a polypeptidewhich comprises growing the host cell which comprises the recombinantexpression vector which comprises the DNA encoding the gE glycoproteinof infectious bovine rhinotracheitis virus under conditions such thatthe recombinant expression vector expresses gE glycoprotein andrecovering the gE glycoprotein so expressed.

The subject invention also provides an antibody directed to an epitopeof the purified gE glycoprotein of infectious bovine rhinotracheitisvirus encoded by the DNA encoding the gE glycoprotein of infectiousbovine rhinotracheitis virus. The antibody may be a monoclonal antibody.

The subject invention also provides a method of detecting the presenceor absence of gE glycoprotein of infectious bovine rhinotracheitis virusin a sample which comprises contacting the sample with an antibodydirected to an epitope of the purified gE glycoprotein of infectiousbovine rhinotracheitis virus encoded by the DNA encoding the gEglycoprotein of infectious bovine rhinotracheitis virus under conditionssuch that the antibody forms a complex with any gE glycoprotein presentin the sample and detecting the presence or absence of such complex. Thesample may be bovine-derived.

The subject invention also provides a method of producing a fetal-safe,live recombinant infectious bovine rhinotracheitis virus which comprisestreating viral DNA from a naturally-occurring live infectious bovinerhinotracheitis virus so as to delete from the virus DNA correspondingto the US2 region of the naturally-occurring infectious bovinerhinotracheitis virus.

The subject invention also provides isolated DNA encoding the US2 geneof an infectious bovine rhinotracheitis virus. The present inventionfurther provides a homology vector for producing a recombinantinfectious bovine rhinotracheitis virus by inserting foreign DNA intothe genomic DNA of an infectious bovine rhinotracheitis virus whichcomprises a double-stranded DNA molecule consisting essentially ofdouble-stranded foreign DNA encoding RNA which does not naturally occurin an animal into which the recombinant infectious bovinerhinotracheitis is introduced, with at one end of the foreign DNA,double-stranded infectious bovine rhinotracheitis viral DNA homologousto genomic DNA located at one side of a site on the genomic DNA which isnot essential for replication of the infectious bovine rhinotracheitisvirus and at the other end of the foreign DNA, double-strandedinfectious bovine rhinotracheitis viral DNA homologous to genomic DNAlocated at the other side of the same site on the genomic DNA. Thedouble-stranded foreign DNA may further comprise a promoter. Thepromoter can be from HSV-1 ICP4, Human cytomegalovirus immediate earlygene or pseudorabies virus glycoprotein X gene. The double-strandedforeign DNA may further comprise a polyadenylation signal. Thepolyadenylation signal may be from HSV-1 thymidine kinase gene orpseudorabies virus glycoprotein X gene. The subject invention alsoprovides a homology vector wherein the RNA encodes a polypeptide. Thepolypeptide may be a detectable marker such as Escherichia coliβ-galactosidase or bacterial transposon neomycin resistance protein. TheDNA which encodes the polypeptide may be flanked on each side byrestriction sites permitting said DNA to be cut out with a restrictionendonuclease which cuts at a limited number of sites on the genome. Thesubject invention further provides for a homology vector wherein theupstream double-stranded infectious bovine rhinotracheitis viral DNA ishomologous to genomic DNA present within the approximately 860 bp NcoIto BamHI subfragment of the HindIII A fragment of infectious bovinerhinotracheitis virus and the downstream double-stranded infectiousbovine rhinotracheitis viral DNA is homologous to genomic DNA presentwithin the approximately 1741 bp BglII to StuI subfragment of theHindIII A fragment of infectious bovine rhinotracheitis virus.

The subject invention further provides a homology vector whereinupstream double-stranded foreign DNA which comprises a promoter anddownstream double-stranded foreign DNA which comprises a polyadenylationsignal flank on each side double-stranded foreign DNA which encodes adetectable marker. The invention further a homology vector wherein theupstream promoter is homologous to genomic DNA present within theapproximately 490 bp PvuII to BamHI subfragment of the BamHI N fragmentof HSV-1 and the downstream polyadenylation signal is homologous togenomic DNA present within the approximately 784 bp SmaI to SmaIsubfragment of the BamHI Q fragment of HSV-1. The invention furtherprovides a homology vector wherein the DNA which encodes a detectablemarker is homologous to the approximately 1541 bp BglII to BamHIfragment of Tn5.

The subject invention also provides a homology vector wherein theupstream double-stranded infectious bovine rhinotracheitis viral DNA ishomologous to genomic DNA present within the approximately 3593 bpHindIII to XhoI subfragment of the HindIII K fragment of infectiousbovine rhinotracheitis virus and the downstream double-strandedinfectious bovine rhinotracheitis viral DNA is homologous to genomic DNApresent within the approximately 785 bp XhoI to NdeI subfragment of theHindIII K fragment of infectious bovine rhinotracheitis virus. Theinvention further provides a homology vector wherein upstreamdouble-stranded foreign DNA which comprises a promoter and downstreamdouble-stranded foreign DNA which comprises a polyadenylation signalflank on each side double-stranded foreign DNA which encodes adetectable marker. This upstream promoter is homologous to genomic DNApresent within the approximately 1191 bp AvaII to PstI subfragment ofthe XbaI B fragment of HCMV and the downstream polyadenylation sequenceis homologous to genomic DNA present within the approximately 753 bpSalI to NdeI subfragment of the BamHI #7 fragment of PRV. The DNA whichencodes a detectable marker is homologous to the approximately 3347 bpBalI to BamHI fragment of pJF751.

The invention further provides a homology vector wherein the upstreamdouble-stranded infectious bovine rhinotracheitis viral DNA ishomologous to genomic DNA present within the approximately 888 bp MluIto SmaI subfragment of the HindIII K fragment of infectious bovinerhinotracheitis virus and the downstream double-stranded infectiousbovine rhinotracheitis viral DNA is homologous to genomic DNA presentwithin the approximately 785 bp XhoI to NdeI subfragment of the HindIIIK fragment of infectious bovine rhinotracheitis virus. The upstreamdouble-stranded foreign DNA may comprise a promoter and double-strandedforeign DNA which comprise a polyadenylation signal flank on each sidedouble-stranded foreign DNA which encodes a detectable marker. Thesubject invention also provides a homology vector wherein the upstreampromoter is homologous to genomic DNA present within the approximately1191 bp AvaII to PstI subfragment of the XbaI B fragment of HCMV and thedownstream polyadenylation signal is homologous to genomic DNA presentwithin the approximately 753 bp SalI to NdeI subfragment of the BamHI #7fragment of PRV. The DNA which encodes a detectable marker is homologousto the approximately 3347 bp BalI to BamHI fragment of pJF571.

The present invention further provides a homology vector wherein theupstream double-stranded infectious bovine rhinotracheitis viral DNA ishomologous to genomic DNA present within the approximately 1704 bp SmaIto SmaI subfragment of the HindIII K fragment of infectious bovinerhinotracheitis virus and the downstream double-stranded infectiousbovine rhinotracheitis viral DNA is homologous to genomic DNA presentwithin the approximately 742 bp NheI to BglI subfragment of the SmaI2.5KB fragment of infectious bovine rhinotracheitis virus. The presentinvention further provides a homology vector wherein upstreamdouble-stranded foreign DNA which comprises a promoter and downstreamdouble-stranded foreign DNA which comprises a polyadenylation signalflank on each side double-stranded foreign DNA which encodes adetectable marker. The upstream promoter is homologous to genomic DNApresent within the approximately 413 bp SalI to BamHI subfragment of theBamHI #10 fragment of PRV and the downstream polyadenylation signal ishomologous to genomic DNA present within the approximately 754 bp NdeIto SalI subfragment of the BamHI #7 fragment of PRV. The detectablemarker is homologous to the approximately 3010 bp BamHI to PvuIIfragment of pJF751.

The present invention provides for a homology vector for producing arecombinant infectious bovine rhinotracheitis virus by deleting DNAwhich encodes a detectable marker which had been inserted into thegenomic DNA of an infectious bovine rhinotracheitis virus comprising adouble-stranded DNA molecule consisting essentially of double-strandedinfectious bovine rhinotracheitis viral DNA homologous to the genomicDNA which flank on each side the DNA to be deleted. The subjectinvention further provides a homology vector wherein the upstreamdouble-stranded infectious bovine rhinotracheitis viral DNA ishomologous to genomic DNA present within the approximately 888 bp MluIto SmaI subfragment of the HindIII K fragment of infectious bovinerhinotracheitis virus and the downstream double-stranded infectiousbovine rhinotracheitis viral DNA is homologous to genomic DNA presentwithin the approximately 785 bp XhoI to NdeI subfragment of the HindIIIK fragment of infectious bovine rhinotracheitis virus.

In a preferred embodiment the homology vectors are designated 691-096.2,756-11.17, and 769-73.3.

The present invention also provides a method of immunizing an animalagainst infectious bovine rhinotracheitis virus which comprisesadministering to the animal an effective immunizing dose of any of thevaccines of the present invention. The animal may be a bovine. Thesubject invention also provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus from which at least a portion ofboth repeat sequences have been deleted, specifically, wherein DNAencoding one or more EcoRV restriction sites has been deleted, andwherein foreign DNA has been inserted into the DNA of the recombinantvirus. The foreign DNA may be a DNA sequence which encodes bovine viraldiarrhea virus glycoprotein g53. The subject invention provides anexample of such a recombinant infectious bovine rhinotracheitis virus,designated S-IBR-032.

The subject invention provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringinfectious bovine rhinotracheitis virus from which DNA from the US2gene, the gE glycoprotein gene and the gG glycoprotein gene have beendeleted so that upon replication, the recombinant infectious bovinerhinotracheitis virus produces no gE glycoprotein and no gGglycoprotein. A Foreign DNA sequence may be inserted in place of thedeleted DNA which encodes gE glycoprotein. The foreign DNA sequence thatmay be inserted can be a foreign DNA sequence which encodes Escherichiacoli β-galactosidase. The subject invention provides an example of sucha recombinant virus, designated S-IBR-039.

The subject invention further provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringIBR virus in which DNA from the US2, gE glycoprotein gene, the gGglycoprotein gene and the thymidine kinase gene has been deleted so thatupon replication, the recombinant infectious bovine rhinotracheitisvirus produces no gE glycoprotein, no gG glycoprotein and no thymidinekinase. The subject invention provides an example of such a recombinantvirus, designated S-IBR-045. A foreign DNA sequence may be inserted inplace of the deleted DNA encoding gE glycoprotein. The foreign DNAsequence may encode Escherichia coli β-galactosidase. The subjectinvention provides an example of such a recombinant virus, designatedS-IBR-044. The foreign DNA sequence may encode bovine viral diarrheavirus g53 glycoprotein. The subject invention provides an example ofsuch a recombinant virus, designated S-IBR-046. The foreign DNA sequencemay encode Parainfluenza virus type 3 fusion protein and Parainfluenzavirus type 3 hemagglutinin protein. The subject application provides anexample of such a virus, designated S-IBR-047. The foreign DNA sequencemay encode Bovine respiratory syncytial virus fusion protein, Bovinerespiratory syncytial virus attachment protein and Bovine respiratorysyncytial virus nucleocapsid protein. The subject invention provides anexample of such a recombinant virus, designated S-IBR-049. The foreignDNA sequence may encode Pasteurella haemolytica leukotoxin andPasteurella haemolytica iron regulated outer membrane proteins. Thesubject invention provides an example of such a recombinant virus,designated S-IBR-051.

The subject invention also provides a recombinant infectious bovinerhinotracheitis virus comprising viral DNA from a naturally-occurringIBR virus from which DNA from the US2 gene, the gE glycoprotein gene,the gG glycoprotein gene and the thymidine kinase gene have been deletedso that upon replication, the recombinant IBR virus produces no gEglycoprotein, no gG glycoprotein and no thymidine kinase. The subjectinvention provides for a foreign DNA sequence inserted in place of theDNA which encodes thymidine kinase. The foreign DNA sequence may encodeEscherichia coli β-glucuronidase. The present invention further providesa recombinant virus wherein a foreign DNA sequence is inserted in placeof the DNA encoding gE glycoprotein. The foreign DNA sequence may encodeEscherichia coli β-galactosidase. The present invention further providesan example of such a recombinant virus, designated S-IBR-043.

The subject invention also provides a vaccine which comprises aneffective immunizing amount of any of the recombinant viruses of thepresent invention and a suitable carrier. The vaccine may contain eitherinactivated or live recombinant virus.

The present invention provides a vaccine which comprises an effectiveimmunizing amount of recombinant virus protective against bovinerespiratory disease complex and a suitable carrier. A recombinant virusmay be a recombinant IBR virus and the recombinant virus can consistessentially of any or all of the recombinant viruses of the presentinvention.

The subject invention also provides for a vaccine which comprises aneffective immunizing amount of a recombinant virus and non-recombinantvirus protective against bovine respiratory disease complex and asuitable carrier.

The subject invention further provides a vaccine which comprises aneffective immunizing amount of a recombinant IBR virus andnon-recombinant virus protective against bovine respiratory diseasecomplex and a suitable carrier. The recombinant IBR virus can consistessentially of any or all of the recombinant viruses of the subjectinvention.

For purposes of this invention, the infectious diseases that contributeto bovine respiratory disease complex include infectious bovinerhinotracheitis, parainfluenza type 3 virus, bovine viral diarrheavirus, bovine respiratory syncytial virus and Pasteurella haemolytica.

For purposes of the present invention, non-recombinant viruses caninclude, but are not limited to, conventionally derived viruses whichinclude killed virus, inactivated bacterins, and modified live viruses.

The subject invention further provides for a method of immunizing ananimal against infectious bovine rhinotracheitis which comprisesadministering to the animal an immunizing dose of any of the vaccines ofthe present invention. The subject invention further provides a methodof immunizing an animal against Parainfluenza type 3 which comprisesadministering to the animal an immunizing dose of the vaccine of thepresent invention that contains the IBR virus encoding antigens forParainfluenza type 3 virus. The subject invention further provides amethod of immunizing an animal against bovine viral diarrhea whichcomprises administering to the animal an immunizing dose of the vaccineof the present invention that contains the IBR virus encoding antigensfor bovine viral diarrhea virus. The subject invention further providesa method of immunizing an animal against bovine respiratory syncytialvirus disease which comprises administering to the animal an immunizingdose of the vaccine of the present invention that contains the IBR virusencoding antigens for bovine respiratory syncytial virus. The subjectinvention further provides for a method of immunizing an animal againstPneumonic pasteurellosis which comprises administering to the animal animmunizing dose of the vaccine of the present invention that containsthe IBR virus encoding antigens for Pasteurella haemolytica.

The invention further provides a method of immunizing an animal againstbovine respiratory disease complex which comprises administering to ananimal an immunizing dose of the vaccine containing the recombinant IBRviruses of the present invention or the recombinant viruses of thepresent invention and non-recombinant viruses. For purposes of thisinvention, the animal may be a bovine. The invention further provides amethod for distinguishing an animal vaccinated with a vaccine whichcomprises an effective immunizing amount of a recombinant virus of thepresent invention from an animal infected with a naturally-occurring IBRvirus which comprises analyzing a sample of a body fluid from the animalfor the presence of gE glycoprotein of IBR virus and at least one otherantigen normally expressed in an animal infected by anaturally-occurring IBR virus, identifying antigens which are present inthe body fluid and determining whether gE glycoprotein is present in thebody fluid, the presence of antigens which are normally expressed in ananimal by a naturally-occurring IBR virus and the absence of gEglycoprotein in the body fluid being indicative of an animal vaccinatedwith the vaccine and not infected with a naturally-occurring IBR virus.

The present invention also provides a vaccine which comprises aneffective immunizing amount of the recombinant infectious bovinerhinotracheitis virus S-IBR-052 and a suitable carrier. The vaccine maycontain either inactivated or live infectious bovine rhinotracheitisS-IBR-052.

In general, the vaccine of this invention contains an effectiveimmunizing amount of S-IBR-052 virus from about 10³ to 10⁸ PFU/dose.Preferably, the effective immunizing amount is from about 10⁴ to 10⁷ or10⁴ to 10⁶ PFU/dose for the live vaccine. Preferably, the live vaccineis created by taking tissue culture fluids and adding stabilizing agentssuch as stabilized, hydrolyzed proteins. Preferably, the inactivatedvaccine uses tissue culture fluids directly after inactivation of thevirus.

The present invention also provides a method of immunizing an animal,particularly a bovine, against disease caused by infectious bovinerhinotracheitis virus which comprises administering to the animal aneffective immunizing dose of the vaccine comprising S-IBR-052.

The present invention provides a method of enhancing an immune responsewhich comprises administering to a subject an effective dose of arecombinant infectious bovine rhinotracheitis virus and a suitablecarrier.

The present invention also provides a method for distinguishing ananimal vaccinated with the infectious bovine rhinotracheitis virusS-IBR-052 from an animal infected with naturally-occurring infectiousbovine rhinotracheitis virus. This method comprises analyzing a sampleof a body fluid from the animal for the presence of IBRV gG or gE and atleast one other antigen which is normally expressed in an animalinfected by a naturally-occurring infectious bovine rhinotracheitisvirus and determining whether the antigen and gG or gE are present inthe body fluid. The presence of the antigen and the absence of gG or gEin the body fluid is indicative of an animal vaccinated with the vaccineand not infected with a naturally-occurring infectious bovinerhinotracheitis virus.

The presence of the antigen and of gG or gE in the body fluid may bedetermined by various methods, for example, by detecting in the bodyfluid antibodies specific for the antigen and for gG or gE.

Methods for constructing, selecting and purifying infectious bovinerhinotracheitis viruses, including S-IBR-052, are detailed in theMaterials and Methods section which follows. Furthermore, Example 25 ofthe specification contains detailed characterization of recombinantinfectious bovine rhinotracheitis virus S-IBR-052.

EXPERIMENTAL DETAILS Materials and Methods

Preparation of IBR Virus Stock Samples

IBR virus stock samples were prepared by infecting MDBK cells at amultiplicity of infection of 0.01 PFU/cell in Dulbecco's Modified EagleMedium (DMEM) containing 2 mM glutamine, 100 units/ml penicillin, 100units/ml streptomycin (these components were obtained from IrvineScientific or an equivalent supplier, and hereafter are referred to ascomplete DME medium) plus 1% fetal bovine serum. After cytopathic effectwas complete, the medium and cells were harvested and the cells werepelleted at 3000 rpm for 5 minutes in a clinical centrifuge. Cells wereresuspended in {fraction (1/10)} the original volume of medium, and anequal volume of skim milk (9% skim milk powder in H₂O weight/volume) wasadded. The virus sample was frozen at −70° C. The titers were usuallyabout 10⁸ PFU/ml.

Preparation of Herpesvirus DNA

For herpesvirus DNA preparation, a confluent monolayer of cells (MDBKfor IBR virus or Vero for PRV) in a 25 cm² flask or 60 mm petri dish wasinfected with 100 gl of virus sample. After overnight incubation, orwhen the cells were showing 100% cytopathic effect, the cells werescraped into the medium. The cells and medium were centrifuged at 3000rpm for 5 minutes in a clinical centrifuge. The medium was decanted, andthe cell pellet was gently resuspended in 0.5 ml of solution containing0.5% NONIDET P-40™ (NP-40, purchased from Sigma Chemical Co., St. Louis,Mo.). The sample was incubated at room temperature for 10 minutes. Tenμl of a stock solution of RNase A (Sigma) was added (stock was 10 mg/ml,boiled for 10 minutes to inactivate DNAse). The sample was centrifugedto pellet nuclei. The DNA pellet was removed with a pasteur pipette orwooden stick and discarded. The supernatant fluid was decanted into a1.5 ml Eppendorf tube containing 25 μl of 20% sodium dodecyl sulfate(Sigma) and 25 μl proteinase-K (10 mg/ml; Boehringer Mannheim). Thesample was mixed and incubated at 37° C. for 30-60 minutes. An equalvolume of water-saturated phenol was added and the sample was mixedbriefly. The sample was centrifuged in an Eppendorf minifuge for 5minutes at full speed. The upper aqueous phase was removed to a newEppendorf tube, and two volumes of absolute ethanol were added and thetube put at −20° C. for 30 minutes to precipitate nucleic acid. Thesample was centrifuged in an Eppendorf minifuge for 5 minutes. Thesupernatant was decanted, and the pellet was washed with ˜300 μl of 80%ethanol, followed by centrifugation in an Eppendorf minifuge for 5minutes. The supernatant was decanted, and the pellet was air dried andrehydrated in ˜16 μl H₂O. For the preparation of larger amounts of DNA,the procedure was scaled up to start with a 850 cm² roller bottle ofMDBK cells. The DNA was stored in 0.01 M tris pH 7.5, 1 mM EDTA at 4° C.

Preparation of Herpesvirus Cell Lysates

For cell lysate preparation, serum free medium was used. A confluentmonolayer of cells (MDBK for IER virus or Vero for PRV) in a 25 cm²flask or a 60 mm petri dish was infected with 100 μl of virus sample.After cytopathic effect was complete, the medium and cells wereharvested and the cells were pelleted at 3000 rpm for 5 minutes in aclinical centrifuge. For media samples medium was concentratedapproximately 10-fold by filtration with a centricon-10microconcentrator (Amicon). For cell samples the cell pellet wasresuspended in 250 μl of disruption buffer (2% sodium dodecyl sulfate,2% β-mercaptoethanol). The samples were sonicated for 30 seconds on iceand stored at −20° C.

Western Blotting Procedure

Samples of lysates, controls and protein standards were run on apolyacrylamide gel according to the procedure of Laemmli (2). After gelelectrophoresis the proteins were transferred according to Sambrook(14). The primary antibody was a mouse hyper-immune serum raised againstchemically-synthesized gG peptides (amino acids 232-252 and 267-287)linked to keyhole limpet hemocyanin. The secondary antibody was a goatanti-mouse alkaline phosphatase coupled antibody.

Molecular Biological Techniques

Techniques for the manipulation of bacteria and DNA, including suchprocedures as digestion with restriction endonucleases, gelelectrophoresis, extraction of DNA from gels, ligation, phosphorylationwith kinase, treatment with phosphatase, growth of bacterial cultures,transformation of bacteria with DNA, and other molecular biologicalmethods are described by Maniatis (6). Except as noted, these were usedwith minor variation.

Ligation

DNA was joined together by the action of the enzyme T4 DNA ligase (BRL).Ligation reactions contained various amounts of DNA (from 0.2 to 20 μg),20 mM Tris pH 7.5, 10 mM MgCl₂, 10 mM dithiothreitol (DTT), 200 μM ATPand 20 units T4 DNA ligase in 10-20 μl final reaction volume. Theligation proceeded for 3-16 hours at 15° C.

DNA Sequencing

Sequencing was performed using the BRL Sequenase Kit and ³⁵S-dATP (NEN).Reactions using both the dGTP mixes and the dITP mixes were performed toclarify areas of compression. Alternatively, compressed areas wereresolved on formamide gels. Templates were double-stranded plasmidsubclones or single stranded M13 subclones, and primers were either madeto the vector just outside the insert to be sequenced, or to previouslyobtained sequence. Sequence obtained was assembled and compared usingDnastar software. Manipulation and comparison of sequences obtained wasperformed with Superclone and Supersee programs from Coral Software.

Southern Blotting of DNA

The general procedure for Southern blotting was taken from Maniatis (6).DNA was blotted to nitrocellulose filters and hybridized to appropriate,labeled DNA probes. Probes for southern blots were prepared using eitherthe Nonradioactive DNA Labeling and Detection Kit of Boehringer Mannheimor the nick translation kit of Bethesda Research Laboratories (BRL). Inboth cases the manufacturers' recommended procedures were followed.

DNA Transfection for Generating Recombinant Virus

The method is based upon the calcium phosphate procedure of Graham andVan der Eb (24) with the following modifications. Virus and/or plasmidDNA were diluted to 298 μl in 0.01 M Tris pH 7.5, 1 mM EDTA. Forty μl 2MCaCl₂ was added followed by an equal volume of 2×HEPES buffered saline(10 g N-2-hydroxyethyl piperazine N′-2-ethanesulfonic acid (HEPES), 16 gNaCl, 0.74 g KCl, 0.25 g Na₂HPO₄ .2H₂O, 2 g dextrose per liter H₂O andbuffered with NaOH to pH 7.4). The mixture was then incubated on ice for10 minutes, and then added dropwise to an 80% confluent monolayer ofMDBK or rabbit skin (RS) cells growing in a 60 mm petri dish under 5 mlof medium (DME plus 2% fetal bovine serum). The cells were incubated 4hours at 37° C. in a humidified incubator containing 5% CO₂. The cellswere then washed with three 5 ml aliquots of 1×PBS (1.15 g Na₂HPO₄, 0.2g KH₂PO₄, 0.8 g NaCl, 0.2 g KCl per liter H₂O), and fed with 5 ml ofmedium (DME plus 2% fetal bovine serum). The cells were incubated at 37°C. as above for 3-7 days until cytopathic effect from the virus was50-100%. Virus was harvested as described above for the preparation ofvirus stocks. This stock was referred to as a transfection stock and wassubsequently screened for recombinant virus by the BLUOGAL™ SCREEN FORRECOMBINANT IBR VIRUS.

Homologous Recombination Procedure for Generating RecombinantHerpesvirus

This method relies upon the homologous recombination between herpesvirusDNA and plasmid homology vector DNA which occurs in tissue culture cellsco-transfected with these elements. From 0.1-1.0 μg of plasmid DNAcontaining foreign DNA flanked by appropriate herpesvirus clonedsequences (the homology vector) were mixed with approximately 0.3 μg ofintact herpesvirus DNA. The DNAs were diluted to 298 μl in 0.01 M TrispH 7.5, 1 mM EDTA and transfected into MDBK cells according to the DNATRANSFECTION FOR GENERATING RECOMBINANT VIRUS (see above).

Direct Ligation Procedure for Generating Recombinant Herpesvirus

Rather than using homology vectors and relying upon homologousrecombination to generate recombinant virus, the technique of directligation to engineer herpesviruses was also developed. In this instance,a cloned foreign gene did not require flanking herpesvirus DNA sequencesbut only required that it have restriction sites available to cut outthe foreign gene fragment from the plasmid vector. A compatiblerestriction enzyme was used to cut herpesvirus DNA. A requirement of thetechnique was that the restriction enzyme used to cut the herpesvirusDNA must cut at a limited number of sites. XbaI, which cuts IBR virusDNA in one place was used. EcoRV which cuts IBR virus DNA in two placeswas used. For PRV XbaI and HindIII, both of which cut in two places wasused. Restriction sites previously introduced into herpesviruses byother methods may also be used. The herpesvirus DNA was mixed with a30-fold molar excess of plasmid DNA (typically 5 μg of virus DNA to 10μg of plasmid DNA), and the mixture was cut with the appropriaterestriction enzyme. The DNA mixture was phenol extracted and ethanolprecipitated to remove restriction enzymes, and ligated togetheraccording to the ligation procedure detailed above. The ligated DNAmixture was then resuspended in 298 μl 0.01 M Tris pH 7.5, 1 mM EDTA andtransfected into cells (MDBK or RS for IBR virus and Vero for PRV)according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS (seeabove). The direct ligation procedure may also be used to delete DNAfrom herpesviruses. Non-essential DNA which is flanked by appropriaterestriction enzyme sites may be deleted by digesting the virus DNA withsuch enzymes and religation. The frequency of engineered virusesgenerated by the direct ligation procedure is high enough that screeningcan be accomplished by restriction enzyme analysis of randomly pickedplaques from the transfection stock.

Bluogal™ Screen for Recombinant Herpesvirus

When the E.coli β-galactosidase (lacZ) marker gene was incorporated intoa recombinant virus the plaques containing recombinants were visualizedby a simple assay. The chemical BLUOGAL™ (GIBCO-Bethesda Research Labs)was incorporated (200 μg/ml) into the agarose overlay during the plaqueassay, and plaques that expressed active β-galactosidase turned blue.The blue plaques were then picked onto fresh cells (MDBK for IBR virusand Vero for PRV) and purified by further blue plaque isolations. Inrecombinant virus strategies in which the E.coli β-galactosidase markergene is removed, the assay involves plaque purifying white plaques froma background of parental blue plaques. In both cases viruses weretypically purified with three rounds of plaque purification.

Screen for Recombinant Herpesvirus Expressing Enzymatic Marker Genes

When the E. coli β-galactosidase (lacZ) or β-glucuronidase (uidA) markergene was incorporated into a recombinant virus the plaques containingrecombinants were visualized by a simple assay. The enzymatic substratewas incorporated (300 μg/ml) into the agarose overlay during the plaqueassay. For the lacZ marker gene the substrate BLUOGAL™ (halogenatedindolyl-β-D-galactosidase, Bethesda Research Labs) was used. For theuidA marker gene the substrate X-Glucuro Chx(5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid Cyclohexylammonium salt,Biosynth AG) was used. Plaques that expressed active marker enzymeturned blue. The blue plaques were then picked onto fresh cells andpurified by further blue plaque isolation. In recombinant virusstrategies in which the enzymatic marker gene is removed the assayinvolves plaque purifying white plaques from a background of parentalblue plaques. In both cases viruses were typically purified with threerounds of plaque purification.

Antibody Screen for Recombinant Herpesvirus

A third method for screening the recombinant virus stock was to lookdirectly for the expression of the foreign gene with antibodies.Herpesvirus plaques were spotted and picked by inserting a toothpickthrough the agarose above the plaque and scraping the plaque area on thedish. Viruses were then rinsed from the toothpick by inserting thetoothpick into a well of a 96-well microtiter dish (Falcon Plastics)containing a confluent monolayer of tissue culture cells that had beenwashed 3 times in DME medium without serum. It was important for thevirus to grow without serum at this stage to allow the immunologicalprocedure to work. After cytopathic effect was complete, the plates wereput at −70° C. to freeze and lyse the cells. The medium was thawed, andthe freeze/thaw procedure was repeated a second time. Then 50-100microliters of medium were removed from each well and filtered undervacuum through a nitrocellulose membrane (S&S BA85) using a DotBlot˜apparatus (BRL). The filter blots were soaked in a blocking solution of0.01 M Tris pH 7.5, 0.1 M NaCl, 3% bovine serum albumin at roomtemperature for two hours with shaking. The filter blots were thenplaced in a sealable bag (Sears SEAL-A-MEAL™ or equivalent), and 10 mlsof the blocking solution that contained 10 microliters of antibodyspecific for the foreign protein were added. After overnight incubationat room temperature with shaking, the blot was washed 3 times with 100mls 0.01 M Tris, pH 7.5, 0.1 M NaCl, 0.05% Tween 20 detergent (Sigma).The blot was put in another sealable bag and 10 mls blocking solutioncontaining 10⁶ counts per minute of ¹²⁵I-protein A (New England Nuclear)were added. After allowing the protein A to bind to the antibody for 2hours at room temperature with shaking, the blot was washed as above,dried, and overlayed with an X-ray film and an intensifying screen(Dupont) and autoradiographed for 1-3 days at −70° C. The film wasdeveloped by standard procedures. Virus from the positive wells whichcontained the recombinant virus was further purified.

Selection of G418 Resistant IBR Virus

The antibiotic G418 (GIBCO) has a wide range of inhibitory activity onprotein synthesis. However, recombinant viruses expressing theaminoglycosidase 3′-phosphotransferase, encoded by the NEO gene of thetransposable element Tn5, are resistant to G418. The transfection stocksof recombinant viruses were grown on MDBK cells in the presence of 500μg/ml G418 in complete DME medium plus 1% fetal bovine serum. After oneor two days at 37° C., plaques from the dishes inoculated with thehighest dilution of virus were picked for virus stocks. The selectionwas repeated a second or third time. The virus stocks generated from theG418 selection were tested for NEO gene insertion by the SOUTHERNBLOTTING OF DNA hybridization procedure described above.

Construction of Deletion Viruses

The strategy used to construct deletion viruses involved the use ofeither homologous recombination and/or direct ligation techniques.Initially a virus was constructed via homologous recombination, in whichthe DNA to be deleted was replaced with a marker gene such as E. coliβ-galactosidase (lacZ) or β-glucuronidase (uidA). A second virus wasthen constructed in which the marker gene was deleted either byhomologous recombination or via direct ligation. The advantage of thisstrategy is that both viruses may be purified by the SCREEN FORRECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The firstvirus is purified by picking blue plaques from a white plaquebackground, the second virus is purified by picking white plaques from ablue plaque background.

Several homology vectors were constructed for the purpose of deletingthe gG, gE and Tk gene coding regions. A detailed description of thesehomology vectors follows.

IBR Virus gE Plasmid

A plasmid may be generated for the purpose of constructing a recombinantcloning vector which expresses the IBR virus glycoprotein E (gE). Thisplasmid may be used to insert the IBR virus gE gene into S-PRV-002 (U.S.Pat. No. 4,877,737). The plasmid will contain the gE gene flanked byXbaI restriction sites. When this plasmid is used with S-PRV-002 and therestriction enzyme XbaI according to the DIRECT LIGATION PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS the resulting recombinant willexpress the IBR virus gE. A detailed description of the plasmid is givenin FIGS. 17A-17E. It may be constructed, utilizing standard recombinantDNA techniques (6), by joining restriction fragments from the followingsources. The plasmid vector is derived from an approximately 2999 basepair XbaI to XbaI restriction fragment of a hybrid cloning vectorderived from pSP64 and pSP65 (Promega). The hybrid cloning vector wasconstructed by joining an approximately 1369 base pair PvuI to SmaIfragment from pSP64 with the approximately 1652 base pair PvuI to SmaIfragment from pSP65. Fragment 1 is an approximately 3647 base pair NdeIto HindIII restriction sub-fragment of the IBR virus HindIII restrictionfragment K (7). Fragment 2 is an approximately 832 base pair HindIII toSacI restriction sub-fragment of an IBR virus 2400 base pair SmaIrestriction fragment. This SmaI fragment has been cloned into the SmaIsite of the plasmid pSP64 (Promega). This plasmid is designated PSY1645.PSY1645 was deposited on Jul. 16, 1991 pursuant to the Budapest Treatyon the International Deposit of Microorganisms for the Purposes ofPatent Procedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. 68650. Note that the lacZ marker gene isflanked by XbaI sites located at Junction B and Junction E in thisplasmid permitting the marker gene to be cut out with XbaI.

Purification of IBR Virus gG

gG was purified from the tissue culture medium of infected MDBK cells.Confluent MDBK cells in serum-free medium were infected at amultiplicity of infection equal to 5, with wild-type, Cooper strain ofIBR virus. The cells and media were harvested at approximatelytwenty-two hours after infection, when the cells showed considerablecytopathic effect and the fluids were centrifuged at 5000 rpm for 15minutes.

The supernatant fluid was concentrated approximately 10-fold byultrafiltration through an Amicon ym-30 membrane, and dialyzed against10 mM NaPO₄ pH 7.2. The dialysate was treated for 20 minutes at 0° C.with 70% perchloric acid to a final concentration of 0.2M perchloricacid, then centrifuged at 12,000 rpm for 20 minutes. The supernatantfluid was then dialyzed against 20mM Tris pH 9.5.

The acid-soluble proteins were separated by column chromatography on aDEAE-Sephacel anion exchange column using a liner gradient elution: 0 to100% A to B where A=20 mM Tris pH 9.5 and B=20 mM Tris pH 9.5/800 mMNaCl. The gG eluted at approximately 35-40% B. Peak fractions wereassayed by Western blot using anti gG peptide sera. Reactive fractionswere combined and dialyzed against 5 mM Tris pH 7.0. The sample was thenconcentrated 10-fold by lyophilization and stored at −20° C.

ELISA Assay

A standard enzyme-linked immunosorbent assay (ELISA) protocol was usedto determine the immune status of cattle following vaccination andchallenge.

A purified gG antigen solution (100 μl at 1 ng/μl in PBS) was allowed toabsorb to the wells of microtiter dishes for 18 hours at 4° C. Thecoated wells were rinsed one time with PBS. Wells were blocked by adding250 μl of PBS containing 1% BSA (Sigma) and incubating 1 hour at 37° C.The blocked wells were rinsed one time with PBS containing 0.02% Tween20. 50 μl of test serum (previously diluted 1:2 in PBS containing 1%BSA) were added to the wells and incubated 1 hour at 37° C. Theantiserum was removed and the wells were washed 3 times with PBScontaining 0.02% Tween 20. 50 μl of a solution containing anti-bovineIgG coupled to horseradish peroxidase (diluted 1:500 in PBS containing1% BSA, Kirkegaard and Perry Laboratories, Inc.) was added to visualizethe wells containing antibody against the specific antigen. The solutionwas incubated 1 hour at 37° C., then removed and the wells were washed 3times with PBS containing 0.02% Tween 20. 100 μl of substrate solution(ATBS, Kirkegaard and Perry Laboratories, Inc.) were added to each welland color was allowed to develop for 15 minutes. The reaction wasterminated by addition of 0.1M oxalic acid. The color was read atabsorbance 410 nm on an automatic plate reader.

Procedure for Generating Monoclonal Antibodies

To produce monoclonal antibodies, 8 to 10 week old BALB/c female micewere vaccinated intraperitoneally seven times at two to four weekintervals with 10⁷ PFU of S-PRV-160. Three weeks after the lastvaccination, mice were injected intraperitoneally with 40 μg of purifiedgG. Spleens were removed from the mice three days after the last antigendose.

Splenocytes were fused with mouse NS1/Ag4 plasmacytoma cells by theprocedure modified from Oi and Herzenberg, (39). Splenocytes andplasmacytoma cells were pelleted together by centrifugation at 300×g for10 minutes. One ml of a 50% solution of polyethylene glycol (m.w.1300-1600) was added to the cell pellet with stirring over one minute.Dulbecco's modified Eagles's medium (5 ml) was added to the cells overthree minutes. Cells were pelleted by centrifugation at 300×g for 10minutes and resuspended in medium with 10% fetal bovine serum andcontaining 100 μM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine(HAT). Cells (100 μl) were added to the wells of eight to ten 96-welltissue culture plates containing 100 μl of normal spleen feeder layercells and incubated at 37° C. Cells were fed with fresh HAT medium everythree to four days.

Hybridoma culture supernatants were tested by the ELISA ASSAY in 96-wellmicrotiter plates coated with 100 ng of purified gG. Supernatants fromreactive hybridomas were further analyzed by black-plaque assay and byWestern Blot. Selected hybridomas were cloned twice by limitingdilution. Ascetic fluid was produced by intraperitoneal injection of5×10⁶ hybridoma cells into pristane-treated BALB/c mice.

Method for cDNA Cloning Bovine Rotavirus g38 Gene

The Calf Nebraska strain of bovine rotavirus (USDA) was propagated onMA-104 cells (Rhesus monkey kidney cells from MA Bioproducts). Confluentmonolayers were infected at a multiplicity of infection of greater than10 in DMEM containing 5 micrograms/ml trypsin. Cells were incubated withvirus for 48 hours or until a cytopathic effect was obtained. Media andcell debris were collected and centrifuged at 10,000×g for 20 minutes at4° C. The supernatant containing the rotavirus was then centrifuged at10,000×g in a preparative Beckman Ti45 rotor at 4° C. Virus pellets wereresuspended in SM medium (50 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl)and homogenized lightly in a Dounce-type homogenizer. The resuspendedvirus was centrifuged at 10,000×g for 10 minutes then loaded onto 25-50%CsCl gradients in SM buffer. Gradients were centrifuged at 100,000×g for4 hours at 20° C. The two blue-white bands representing intact virionsand cores of rotavirus were collected, diluted, and the CsCl gradientprocedure was repeated a second time. Virus obtained from the secondgradient was dialyzed overnight against SM buffer at 4° C.

Dialyzed bovine rotavirus was twice extracted with an equal volume ofSDS/phenol, then twice more with chloroform:isoamylalcohol (24:1). Thedouble stranded RNA was precipitated with ethanol in the presence of 0.2M sodium acetate, centrifuged and resuspended in water. The yield wastypically 100 micrograms from 1,000 cm² of infected cells.

160 micrograms of double-stranded bovine rotavirus RNA obtained from theabove procedure was mixed with one microgram each of two syntheticoligonucleotide primers in a volume of 160 microliter (sequences ofprimers were: 5′-GGGAATTCTGCAGGTCACATCATACAATTCTAATCTAAG-3′ (SEQ IDNO: 1) and 5′-GGGAATTCTGCAGGCTTTAAAAGAGAGAATTTCCGTTTGGCTA-3′ (SEQ ID NO:2) derived from the published sequence of bovine rotavirus (40). TheRNA-primer mixture was boiled for 3 minutes in a water bath then chilledon ice. Additions of 25 microliters of 1 M Tris-HCl pH 8.3, 35microliters of 1 M KCl, 10 microliters of 0.25 M MgCl₂, 7 microliters of0.7 M 2-mercaptoethanol, 7 microliters of 20 mM dNTP's, and 6microliters of reverse transcriptase (100 units) were made sequentially.The reaction was incubated at 42° C. for 1.5 hours then 10 microlitersof 0.5 M EDTA pH 8.0 was added and the solution was extracted once withchloroform:phenol (1:1). The aqueous layer was removed and to it 250microliters of 4 M ammonium acetate and 1.0 ml of 95% ethanol was added,the mixture was frozen in dry ice and centrifuged in the cold. Theresulting pellet was resuspended in 100 microliters of 10 mM Tris-HCl pH7.5 and the ammonium acetate precipitation procedure was repeated. Thepellet was resuspended in 100 microliters of 0.3 M KOH and incubated atroom temperature overnight, then at 37° C. for 2 hours. The solution wasbrought to neutral pH by addition of 10 microliters of 3.0 M HCl and 25microliters of 1.0 M Tris-HCl pH 7.5 The resulting single-stranded cDNAwas then precipitated two times by the above-described ammoniumacetate-ethanol procedure. The pellet obtained was resuspended in 50microliters of 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, boiled ina water bath for 2 minutes, then incubated at 59° C. for 16 hours. Thesolution was lyophilized to a volume of 15 microliters and the resultingdouble-stranded cDNA was run on a 1.0% agarose gel (Sigma agarose TypeII). The ethidium bromide-stained DNA migrating at 1,000-1,100 base pairlength was excised from the gel and electroeluted in a CBS electroeluterdevice. The solution was lyophilized, and the cDNA was resuspended in 25microliters of water. To this solution was added 2 microliters of 1.0 MTris-HCl pH 7.5, 2 microliters of 1 M KCl, 1 microliter of 0.25 M MgCl₂,1 microliter of 20 mM dNTP's and 5 units of E. coli DNA polymerase I.The reaction was incubated at room temperature for 15 minutes, thenchloroform/phenol extracted and ammonium acetate-ethanol precipitated asdescribed above. The resulting cDNA was tailed with dCTP using terminaldeoxynucleotide transferase (BRL buffer and enzyme used). The reactionwas stopped with 2 microliters of 0.5 M EDTA, chloroform/phenolextracted and precipitated with sodium acetate in the presence of 10micrograms of carrier tRNA. The resuspended cDNA was mixed with 200 ngof dGMP-tailed Pst I cut pBR322 (BRL catalog #5355SA) in 200 microlitersof 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, heated to 65° C. for 5minutes, then 57° C. for 2 hours. The annealed cDNA-vector pBR322 wastransformed onto E. coli DH-1 cells prepared for high efficiencytransformation. Colonies that showed sensitivity to ampicillin andtetracycline resistance were grown and DNA was prepared and cut with PstI to determine the size of the cDNA insert. Several clones having Pst Iinserts of 1,050-1,100 base pairs were analyzed and found to haveidentical restriction enzyme digest patterns. For one of these clones,the 1,100 base pair PstI insert was subcloned into a M13 phagesequencing vector. Part of the DNA sequence of this clone was determinedand was found to be identical to the published sequence (40).

CDNA Cloning

cDNA cloning refers to the methods used to convert RNA molecules intoDNA molecules following state of the art procedures. Applicants' methodsare described in Gubler and Hoffman (23). Bethesda Research Laboratories(Gaithersburg, Md.) have designed a cDNA Cloning Kit that is verysimilar to the procedures used by applicants and contains the best setof reagents and protocols to duplicate the results.

For cloning virus mRNA species, a host cell line sensitive to infectionby the virus was infected at 5-10 plaque forming units per cell. Whencytopathic effect was evident, but before total destruction, the mediumwas removed and the cells were lysed in 10 mls lysis buffer (4 Mguanidine thiocyanate, 0.1% antifoam A, 25 mM sodium citrate pH 7.0,0.5% N-lauroyl sarcosine, 0.1 M beta-mercaptoethanol). The cell lysatewas poured into a sterilized Dounce homogenizer and homogenized on ice8-10 times until the solution was homogenous. For RNA purification, 8mls of cell lysate were gently layered over 3.5 mls of CsCl solution(5.7 M CsCl, 25 mM sodium citrate pH 7.0) in a Beckman SW41 centrifugetube. The samples were centrifuged for 18 hours at 20° C. at 36,000 rpmin a Beckman SW41 rotor. The tubes were put on ice and the supernatantsfrom the tubes were carefully removed by aspiration to leave the RNApellet undisturbed. The pellet was resuspended in 400 microliters glassdistilled water, and 2.6 mls of guanidine solution (7.5 M guanidine-HCl,25 mM sodium citrate pH 7.0, 5 mM dithiothreitol) were added. Then 0.37volumes of 1 M acetic acid were added, followed by 0.75 volumes of coldethanol and the sample was put at −20° C. for 18 hours to precipitateRNA. The precipitate was collected by centrifugation in a Sorvallcentrifuge for 10 min at 4° C. at 10,000 rpm in an SS34 rotor. Thepellet was dissolved in 1.0 ml distilled water, recentrifuged at 13,000rpm, and the supernatant saved. RNA was reextracted from the pellet 2more times as above with 0.5 ml distilled water, and the supernatantswere pooled. A 0.1 volume of 2 M potassium acetate solution was added tothe sample followed by 2 volumes of cold ethanol and the sample was putat −20° C. for 18 hours. The precipitated RNA was collected bycentrifugation in the SS34 rotor at 4° C. for 10 minutes at 10,000 rpm.The pellet was dissolved in 1 ml distilled water and the concentrationtaken by absorption at A260/280. The RNA was stored at −70° C.

mRNA containing polyadenylate tails (poly-A) was selected using oligo-dTcellulose (Pharmacia #27 5543-0). Three milligrams of total RNA wasboiled and chilled and applied to a 100 mg oligo-dT cellulose column inbinding buffer (0.1 M Tris pH 7.5, 0.5 M LiCl, 5 mM EDTA pH 8.0, 0.1%lithium dodecyl sulfate). The retained poly-A⁺ RNA was eluted from thecolumn with elution buffer (5 mM Tris pH 7.5, 1 mM EDTA pH 8.0, 0.1%sodium dodecyl sulfate). This mRNA was reapplied to an oligo-dT columnin binding buffer and eluted again in elution buffer. The sample wasprecipitated with 200 mM sodium acetate and 2 volumes cold ethanol at−20° C. for 18 hours. The RNA was resuspended in 50 microlitersdistilled water.

Ten micrograms poly-A⁺ RNA was denatured in 20 mM methyl mercuryhydroxide for 6 minutes at 22° C. Beta-mercaptoethanol was added to 75mM and the sample was incubated for 5 min at 22° C. The reaction mixturefor first strand cDNA synthesis in 0.25 ml contained 1 microgramoligo-dT primer (P-L Biochemicals) or 1 microgram synthetic primer, 28units placental ribonuclease inhibitor (Bethesda Research Labs #5518SA),100 mM Tris pH 8.3, 140 mM KCl, 10 mM MgCl₂, 0.8 mM dATP, dCTP, dGTP,and dTTP (Pharmacia), 100 microcuries ³²P-labelled dCTP (New EnglandNuclear #NEG-013H), and 180 units AMV reverse transcriptase (MolecularGenetics Resources #MG 101). The reaction was incubated at 42° C. for 90minutes, and then was terminated with 20 mM EDTA pH 8.0. The sample wasextracted with an equal volume of phenol/chloroform (1:1) andprecipitated with 2 M ammonium acetate and 2 volumes of cold ethanol−20° C. for 3 hours. After precipitation and centrifugation, the pelletwas dissolved in 100 microliters distilled water. The sample was loadedonto a 15 ml G-100 Sephadex column (Pharmacia) in buffer (100 mM Tris pH7.5, 1 mM EDTA pH 8.90, 100 mM NaCl). The leading edge of the eluted DNAfractions were pooled, and DNA was concentrated by lyophilization untilthe volume was about 100 microliters, then the DNA was precipitated withammonium acetate plus ethanol as above.

The entire first strand sample was used for second strand reaction whichfollow the Gubler and Hoffman (23) method except that 50 micrograms/mldNTP's, 5.4 units DNA polymerase I (Boehringer Mannheim #642-711), and100 units/ml E. coli DNA ligase (New England Biolabs #205) in a totalvolume of 50 microliters were used. After second strand synthesis, thecDNA was phenol/chloroform extracted and precipitated. The DNA wasresuspended in 10 microliters distilled water, treated with 1 microgramRNase A for 10 minutes at 22° C., and electrophoresed through a 1%agarose gel (Sigma Type II agarose) in 40 mM Tris-acetate buffer pH6.85. The gel was stained with ethidium bromide, and DNA in the expectedsize range was excised from the gel and electroeluted in 8 mMTris-acetate pH 6.85. Electroeluted DNA was lyophilized to about 100microliters, and precipitated with ammonium acetate and ethanol asabove. The DNA was resuspended in 20 microliters water.

Oligo-dC tails were added to the DNA to facilitate cloning. The reactioncontained the DNA, 100 mM potassium cacodylate pH 7.2, 0.2 mMdithiothreitol, 2 mM CaCl₂, 80 micromoles dCTP, and 25 units terminaldeoxynucleotidyl transferase (Molecular Genetic Resources #S1001) in 50microliters. After 30 minutes at 37° C., the reaction was terminatedwith 10 mM EDTA, and the sample was phenol/chloroform extracted andprecipitated as above.

The dc-tailed DNA sample was annealed to 200 ng plasmid vector pBR322that contained oligo-dG tails (Bethesda Research Labs #5355 SA/SB) in200 microliters of 0.01 M Tris pH 7.5, 0.1 M NaCl, 1 mM EDTA pH 8.0 at65° C. for 2 minutes and then 57° C. for 2 hours. Fresh competent E.coli DH-1 cells were prepared and transformed as described by Hanahan(41) using half the annealed cDNA sample in twenty 200 microliteraliquots of cells. Transformed cells were plated on L-broth agar platesplus 10 micrograms/ml tetracycline. Colonies were screened for thepresence of inserts into the ampicillin gene using Ampscreen (BethesdaResearch Labs #5537 UA), and the positive colonies were picked foranalysis.

Polymerase Fill-in Reaction

DNA was resuspended in buffer containing 50 mM Tris pH 7.4, 50 mM KCl, 5mM MgCl₂, and 400 micromolar each of the four deoxynucleotides. Tenunits of Klenow DNA polymerase (BRL) were added and the reaction wasallowed to proceed for 15 minutes at room temperature. The DNA was thenphenol extracted and ethanol precipitated as above.

Cloning of Bovine Viral Diarrhea Virus g53 and g48 Genes

The bovine viral diarrhea (BVDV) g53 gene was cloned essentially asdescribed earlier (see CDNA CLONING) using the random priming method(6). Viral RNA prepared from BVDV Singer strain grown in MADIN-DARBYbovine kidney (MDBK) cells was converted to cDNA using the randompriming method. The cDNA was used for second strand reaction (23) andthe resulting double stranded DNA was used cloned as described in thecDNA CLONING procedure. From this procedure a series of clones wereobtained that comprised parts of the genome of BVDV. The location of thegene for g53 gene has been published (66) and this sequence informationwas used to locate and isolate the g53 encoding region from the 449kilodalton primary translation product open reading frame contained inthe complete cDNA clone.

The bovine viral diarrhea g48 and g53 genes were cloned by a PCR CLONINGprocedure essentially as described by Katz et al. (J. Virology 64:1808-1811 (1990)) for the HA gene of human influenza. Viral RNA preparedfrom BVD virus Singer strain grown in Madin-Darby bovine kidney (MDBK)cells was first converted to cDNA utilizing an oligonucleotide primerspecific for the target gene. The cDNA was then used as a template forpolymerase chain reaction (PCR) cloning (67) of the targeted region. ThePCR primers were designed to incorporate restriction sites which permitthe cloning of the amplified coding regions into vectors containing theappropriate signals for expression in IBRV. One pair of oligonucleotideswere required for each coding region. The g48 gene coding region (aminoacids 1-226) from the BVDV Singer strain (66) was cloned using thefollowing primers: 5′-ACGTCGGATCCCTTACCAAACCACGTCTTACTCTTGTTTTCC-3′ (SEQID NO: 3) for cDNA priming and combined with5′-ACATAGGATCCCATGGGAGAAAACATAACACAGTGGAACC-3′ (SEQ ID NO: 4) for PCR.The g53 gene coding region (amnio acids 1-394) from the BVDV Singerstrain (66) was cloned using the following primers:5′-CTTGGATCCTCATCCATACTGAGTCCCTGAGGCCTTCTGTTC-3′ (SEQ ID NO: 5) for cDNApriming and combined with 5′-CATAGATCTTGTGGTGCTGTCCGACTTCGCA-3′ (SEQ IDNO: 6) for PCR. Note that this general strategy is used to clone thecoding region of g48 and g53 genes from other strains of BVDV.

Cloning of Bovine Respiratory Syncytial Virus Fusion Protein andNucleocapsid Protein Genes

The bovine respiratory virus (BRSV) fusion (F), attachment (G), annucleocapsid protein (N) genes have been cloned essentially as describedby Katz et al. (Journal of Virology, volume 64, 1808-1811 (1990)) forthe HA gene of human influenza. Viral RNA prepared from virus grown inbovine nasal turbinate (BT) cells was first converted to cDNA utilizingan oligo nucleotide primer specific for the target gene. The cDNA wasthen used as a template for polymerase chain reaction (PCR) cloning (67)of the targeted region. The PCR primers were designed to incorporaterestriction sites which permit the cloning of the amplified codingregions into vectors containing the appropriate signals for expressionin IBRV. One pair of oligo nucleotides was required for each codingregion. The N gene coding region from the BRSV strain 375 (ATCC No. VR1339) was cloned utilizing the following primers:5′-CGTCGGATCCCTCACAGTTCCACATCATTGTCTTTGGGAT-3′ (SEQ ID NO: 7) for cDNApriming and combined with5′-CTTAGGATCCCATGGCTCTTAGCAAGGTCAAACTAAATGAC-3′ (SEQ ID NO: 8) for PCR.The G gene coding region from the BRSV strain 375 (ATCC No. VR 1339) wascloned utilizing the following primers:5′-CGTTGGATCCCTAGATCTGTGTAGTTGATTGATTTGTGTGA-3′ (SEQ ID NO: 9) for cDNApriming and combined with5′-CTCTGGATCCTCATACCCATCATCTTAAATTCAAGACATTA-3′ (SEQ ID NO: 10) for PCR.The F gene from strain 375 (ATCC NO. VR 1339) of BRSV was clonedutilizing the following primers:5′-TGCAGGATCCTCATTTACTAAAGGAAAGATTGTTGAT-3′ (SEQ ID NO: 11) for cDNApriming and combined with 5′-CTCTGGATCCTACAGCCATGAGGATGATCATCAGC-3′ (SEQID NO: 12) for PCR. Note that this general strategy may be used to clonethe coding region of F and N genes from other strains of BRSV.

Cloning of Parainfluenza-3 Virus Fusion and Hemagglutinin Genes

The parainfluenza-3 virus fusion (F) and hemagglutinin (HN) genes werecloned by a cDNA CLONING procedure as described in Examples 16 and 17and also by a PCR CLONING procedure essentially as described by Katz etal. (Journal of Virology, volume 64, 1808-1811 (1990)) for the HA geneof human influenza. Viral RNA prepared from virus grown in Madin-Darbybovine kidney (MDBK) cells was first converted to cDNA utilizing anoligonucleotide primer specific for the target gene. The cDNA was thenused as a template for polymerase chain reaction (PCR) cloning (67) ofthe targeted region. The PCR primers were designed to incorporaterestriction sites which permit the cloning of the amplified codingregions into vectors containing the appropriate signals for expressionin IBRV. One pair of oligonucleotides were required for each codingregion. The F gene coding region from the PI-3 strain SF-4 (VR-281) wascloned using the following primers:5′-TTATGGATCCTGCTGCTGTGTTGAACAACTTTGT-3′ (SEQ ID NO: 13) for CDNApriming combined with 5′-CCGCGGATCCCATGACCATCACAACCATAATCATAGCC-3′ (SEQID NO: 14) for PCR. The HN gene coding region from PI-3 strain SF-4(VR-281) was cloned utilizing the following primers:5′-CGTCGGATCCCTTAGCTGCAGTTTTTTGGAACTTCTGTTTTGA-3′ (SEQ ID NO: 15) forcDNA priming and combined with5′-CATAGGATCCCATGGAATATTGGAAACACACAAACAGCAC-3′ (SEQ ID NO: 16) for PCR.Note that this general strategy is used to clone the coding region of Fand HN genes from other strains of PI-3.

Cloning of Pasteurella Haemolytica Leukotoxin and Iron Regulated OuterMembrane Protein(S)

The Pasteurella haemolytica strain A1 leukotoxin gene was cloned from agenomic DNA sample. Genomic DNA was prepared from P. haemolytica A1cells grown in culture (68) by the methods described in Maniatis et al.(1982). The purified P. haemolytica DNA was then used as a template forpolymerase chain reaction (PCR) cloning (67) of the targeted leukotoxingene. The PCR primers were designed so that restriction endonucleasesites were incorporated that allow the cloning of the 102 kilodaltontoxin portion of the gene into vectors containing the appropriatesignals for expression in IBR. The P. haemolytica A1 (ATTC 43279 biotypeA, serotype 1) leukotoxin gene was cloned utilizing the followingprimers: 5′-TATAGATCTTAGACTTACAACCCTAAAAAAC-3′ (SEQ ID NO: 17) and5′-CGTGGATCCAACTCTATAATGTGTGAAACAATATAG-3′ (SEQ ID NO: 18) for PCR. Notethat this general strategy is used to clone the coding regions for theleukotoxin gene of all P. haemolytica serotypes.

The P. haemolytica A1 iron regulated outer membrane proteins (IRP) of 3major polypeptides with molecular weights of 35, 70 and 100 kilodaltons.The DNA coding for the array of P. haemolytica genes can be cloned inEscherichia coli using plasmid vectors essentially as described inManiatis et al. (1982). The clone library is constructed by partialdigestion of the genomic DNA. The IRP genes can be isolated from thislibrary of P. haemolytica clones by screening for the production of ironregulated outer membrane antigens by a colony enzyme-linkedimmunosorbent assay blot method with antiserum that is specific to theIRPs. This antiserum may be obtained by eluting antibodies derived frompolyclonal antiserum raised against whole P. haemolytica or membraneenriched fractions but selectively bound to the IRPs on Western blots(69). The specificity of the antibodies can be verified by immunoblotscreening of P. haemolytica polypeptides from iron restricted and ironinduced cultures.

Vaccination Studies in Calves with Inactivated IBR Virus

Calves, seronegative to IBR virus, were housed in facilities secure fromIBR virus exposure. Groups of four calves were vaccinatedintramuscularly with vaccines containing 10^(7.3) or 10^(8.0) plaqueforming units of inactivated IBR virus formulated with an oil adjuvant.A second vaccination was given 21 days later; four calves weremaintained as unvaccinated controls. At 21 days after the secondvaccination, animals were challenged intranasally with virulentwild-type IBR virus. After vaccination and challenge, animals wereobserved and the injection site was palpated weekly. Blood samples weretaken on days 0, 7, 21, 28, and 42 post vaccination. After challenge,animals were observed daily for clinical signs of IBR. Blood sampleswere taken on days 7 and 13 post challenge. Nasal swabs were collectedon days 3, 6, 9, and 12 post challenge.

Homology Vector 129-71.5

The plasmid 129-71.5 was constructed for the purpose of deleting aportion of the TK gene coding region from the IBR virus. It incorporatesa selectable marker, the bacterial transposon neomycin resistance gene,flanked by IBR virus DNA. Upstream of the marker gene is anapproximately 860 base pair fragment of IBR virus DNA which ends withsequences encoding amino acids 1-62 of the TK primary translationproduct. Downstream of the marker gene is an approximately 1741 basepair fragment of IBR virus DNA which begins with sequences encodingamino acids 156-367 of the TK primary translation product. When thisplasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS, it will replace the DNA coding foramino acids 63-155 of the TK primary translation product with DNA codingfor the marker gene. Note that the marker gene will be under the controlof the herpes simplex type 1 alpha-4 immediate early gene promoter (5).A detailed description of the plasmid is given in FIGS. 7A-7H. It wasconstructed from the indicated DNA sources utilizing standardrecombinant DNA techniques (6). It may be constructed by joiningrestriction fragments from the following sources with the synthetic DNAsequences indicated in FIGS. 7A-7H. The plasmid vector is derived froman approximately 2975 base pair SmaI to HindIII restriction fragment ofpSP65 (Promega). Fragment 1 is an approximately 860 base pair NcoI toBamHI restriction fragment of the IBR virus HindIII restriction fragmentA (7). This fragment is located on an approximately 5500 base pair ClaIto NruI fragment contained in the IBR virus HindIII A fragment. Fragment2 is an approximately 490 base pair PvuII to BamHI restrictionsub-fragment of the HSV-1 BamHI restriction fragment N (5). Note thatthe HSV-1 oriS region has been removed from this fragment by deletion ofthe sequences between the SmaI sites located 1483 and 128 base pairsaway from the PvuII end (10). Fragment 3 is an approximately 1541 basepair BglII to BamHI restriction fragment of plasmid pNEO (P.L.Biochemicals, Inc.). Fragment 4 is an approximately 784 base pair SmaIto SmaI restriction sub-fragment of the HSV-1 BamHI restriction fragmentQ (10). Note that this fragment is oriented such that thepolyadenylation sequence (AATAAA) is located closest to junction D.Fragment 5 is an approximately 1741 base pair BglII to StuI restrictionsub-fragment from the IBR HindIII restriction fragment A (7).

Plasmid 459-12.6

The plasmid 459-12.6 was generated for the purpose of constructing arecombinant cloning vector which expresses the IBR virus glycoprotein G.This was accomplished by inserting the IBR virus gG gene into S-PRV-013(U.S. Ser. No. 07/823,102 filed Jan. 27, 1986). Plasmid 459-12.6contains a chimeric gene under the control of the IBR virus gG promoter.The chimeric gene expresses a fusion protein consisting of the first 362amino acids of IBR virus gG fused to amino acids 421-467 of the PRV gIII(13) followed by amino acids 480-498 of the PRV gX (12). The chimericgene is flanked by HindIII restriction sites. When this plasmid is usedwith S-PRV-013 and the restriction enzyme HindIII according to theDIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS theresulting recombinant will express the IBR virus gG. A detaileddescription of the plasmid is given in FIGS. 11A-11H. It was constructedfrom the indicated DNA sources utilizing standard recombinant DNAtechniques (6). It may be constructed by joining restriction fragmentsfrom the following sources with the synthetic DNA sequences indicated inFIGS. 11A-11H. The plasmid vector is derived from an approximately 2999base pair XbaI to XbaI restriction fragment of a hybrid cloning vectorderived from pSP64 and pSP65 (Promega). The hybrid cloning vector wasconstructed by joining approximately 1369 base pair PvuI to SmaIfragment from pSP64 with the approximately 1652 base pair PvuI to SmaIfragment from pSP65. Fragment 1 is an approximately 182 base pair PstIto EcoRV restriction sub-fragment of the HCMV XbaI restriction fragmentB (16). Fragment 2 is an approximately 2121 base pair MluI to XhoIrestriction sub-fragment of the IBR virus HindIII restriction fragment K(7) Fragment 3 is an approximately 121 base pair XhoI to BamHIrestriction sub-fragment of the PRV BamHI restriction fragment #2 (3).Fragment 4 is an approximately 760 base pair NdeI to SalI restrictionsub-fragment of the PRV BamHI restriction fragment #7 (3).

Homology Vector 439-01.31

The plasmid 439-01.31 was constructed for the purpose of deleting aportion of the gG gene coding region from the IBR virus. It incorporatesan E.coli β-galactosidase marker gene flanked by IBR virus DNA.Downstream of the marker gene is an approximately 3593 base pairfragment of IBR virus DNA which ends with sequences encoding the first262 amino acids of the gG primary translation product. Upstream of themarker gene is an approximately 785 base pair fragment of IBR virus DNAwhich begins with sequences encoding the last 80 amino acids of the gGprimary translation product. When this plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANTHERPESVIRUS it will replace the DNA coding for amino acids 263-361 ofthe gG primary translation product with DNA coding for the marker gene.Note that the β-galactosidase (lacZ) marker gene will be under thecontrol of the human cytomegalovirus immediate early gene promoter. Adetailed description of the plasmid is given in FIGS. 12A-12H. It wasconstructed from the indicated DNA sources utilizing standardrecombinant DNA techniques (6). It may be constructed by joiningrestriction fragments from the following sources with the synthetic DNAsequences indicated in FIGS. 12A-12H. The plasmid vector is derived froman approximately 2965 base pair HindIII to SmaI restriction fragment ofpSP64 (Promega). Fragment 1 is an approximately 3593 base pair HindIIIto XhoI restriction fragment of the IBR HindIII restriction fragment K(7). Fragment 2 is an approximately 753 base pair SalI to NdeIrestriction fragment of the PRV BamHI restriction fragment #7 (3). Notethat this fragment was resected with Exonuclease III/S1 nucleasedigestion such that approximately 57 base pairs were removed from theNdeI end. Fragment 3 is an approximately 3347 base pair BalI to BamHIrestriction fragment of plasmid pJF751 (38). Fragment 4 is anapproximately 1191 base pair AvaI to PstI restriction fragment from theHCMV XbaI restriction fragment E (16). Fragment 5 is an approximately785 base pair XhoI to NdeI restriction fragment from the IBR HindIIIrestriction fragment K (7). Note that the lacZ marker gene is flanked byXbaI sites located at Junction B and Junction E in this plasmidpermitting the marker gene to be cut out with XbaI.

Homology Vector 439-21.69

The plasmid 439-21.69 was constructed for the purpose of deleting aportion of the gG gene coding region from the IBR virus. It incorporatesan E.coli β-galactosidase (lacZ) marker gene flanked by IBR virus DNA.Downstream of the marker gene is an approximately 888 base pair fragmentof IBR virus DNA which begins approximately 1042 base pairs upstream ofthe initiation codon of the gG gene and ends approximately 154 basepairs upstream of the initiation codon of the gG gene. Upstream of themarker gene is an approximately 785 base pair fragment of IBR virus DNAwhich begins with sequences encoding the last 80 amino acids of the gGprimary translation product. When this plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANTHERPESVIRUS it will replace the DNA coding for amino acids 1-361 of thegG primary translation product with DNA coding for the marker gene. Notethat the β-galactosidase (lacZ) marker gene will be under the control ofthe human cytomegalovirus immediate early gene promoter. A detaileddescription of the plasmid is given in FIGS. 13A-13H. It was constructedfrom the indicated DNA sources utilizing standard recombinant DNAtechniques (6). It may be constructed by joining restriction fragmentsfrom the following sources with the synthetic DNA sequences indicated inFIGS. 13A-13H. The plasmid vector is derived from an approximately 2965base pair HindIII to SmaI restriction fragment of pSP64 (Promega).Fragment 1 is an approximately 3593 base pair HindIII to XhoIrestriction fragment of the IBR HindIII restriction fragment K (7).Fragment 2 is an approximately 753 base pair SalI to NdeI restrictionfragment of the PRV BamHI restriction fragment #7 (3). Note that thisfragment was resected with Exonuclease III/S1 nuclease digestion suchthat approximately 57 base pairs were removed from the NdeI end.Fragment 3 is an approximately 3347 base pair BalI to BamHI restrictionfragment of plasmid pJF751 (38). Fragment 4 is an approximately 1191base pair AvaI to PstI restriction fragment from the HCMV XbaIrestriction fragment E (16). Fragment 5 is an approximately 785 basepair XhoI to NdeI restriction fragment from the IBR HindIII restrictionfragment K (7). Note that the lacZ marker gene is flanked by XbaI siteslocated at Junction B and Junction E in this plasmid permitting themarker gene to be cut out with XbaI.

Homology Vector 439-70.4

The plasmid 439-70.4 was constructed for the purpose of deleting theE.coli β-galactosidase (lacZ) marker gene from S-IBR-035 virus. Itincorporates two regions of IBR viral DNA which flank the marker gene inS-IBR-035. The first region is an approximately 888 base pair fragmentof IBR virus DNA which begins approximately 1042 base pairs upstream ofthe initiation codon of the gG gene and ends approximately 154 basepairs upstream of the initiation codon of the gG gene. The second regionis an approximately 785 base pair fragment of IBR virus DNA which beginswith sequences encoding the last 80 amino acids of the gG primarytranslation product. When this plasmid is used in conjunction withS-IBR-035 DNA according to the HOMOLOGOUS RECOMBINATION PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS it will delete the DNA coding for theE. coli β-galactosidase (lacZ) marker gene. A detailed description ofthe plasmid is given in FIGS. 14A-14E. It was constructed from theindicated DNA sources utilizing standard recombinant DNA techniques (6).It may be constructed by joining restriction fragments from thefollowing sources with the synthetic DNA sequences indicated in FIGS.14A-14E. The plasmid vector is derived from an approximately 2965 basepair HindIII to SmaI restriction fragment of pSP64 (Promega). Fragment 1is an approximately 3593 base pair HindIII to XhoI restriction fragmentof the IBR HindIII restriction fragment K (7). Fragment 2 is anapproximately 785 base pair XhoI to NdeI restriction fragment from theIBR HindIII restriction fragment K (7).

Homology Vector 523-78.72

The plasmid 523-78.72 was constructed for the purpose of deleting aportion of the gE coding region from the IBR virus. It was also used toinsert foreign DNA into IBRV. Plasmid 523-78.72 was constructed bydigestion of the plasmid 536-03.5 with the enzyme XbaI followed byreligation to remove the lacZ marker gene. It incorporates two regionsof IBR viral DNA which flanked the marker gene in S-IBR-039. The firstregion is an approximately 1704 base pair fragment of IBR virus DNAwhich ends with sequences encoding amino acids 1-76 of the gE primarytranslation product. The second region is an approximately 742 base pairfragment of IBR virus DNA which begins with sequences encoding aminoacids 548-617 of the gE primary translation product. When the plasmid523-78.72 is used with S-IBR-039 according to the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS, it willdelete the DNA coding for the E. coli β-galactosidase (lacZ) markergene. A detailed description of plasmid 523-78.72 is given in FIGS.26A-26B. It may be constructed utilizing standard recombinant DNAtechniques (6), by joining restriction fragments from the followingsources with the synthetic DNA sequences indicated in FIGS. 26A-26B. Theplasmid vector 523-78.72 is derived from an approximately 2975 base pairSmaI to HindIII restriction fragment of pSP65 (Promega). Fragment 1 isan approximately 1704 base pair SmaI to SmaI restriction sub-fragment ofthe IBR HindIII restriction fragment K (7). Fragment 2 is anapproximately 742 base pair NheI to BglI sub-fragment of an IBR virus2500 base pair SmaI fragment.

Homology Vector 536-03.5

The plasmid 536-03.5 was constructed for the purpose of deleting aportion of the gE gene coding region from the IBR virus. It incorporatesan E.coli β-galactosidase (lacZ) marker gene flanked by IBR virus DNA.Upstream of the marker gene is an approximately 1704 base pair fragmentof IBR virus DNA which ends with sequences encoding amino acids 1-76 ofthe gE primary translation product. Downstream of the marker gene is anapproximately 742 base pair fragment of IBR virus DNA which begins withsequences encoding amino acids 548-617 of the gE primary translationproduct. When this plasmid is used according to the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS, it willreplace the DNA coding for amino acids 77-547 of the gE primarytranslation product with DNA coding for the marker gene. Note that theβ-galactosidase (lacZ) marker gene will be under the control of the PRVgX. A detailed description of the plasmid is given in FIGS. 18A-18G. Itmay be constructed utilizing standard recombinant DNA techniques (6), byjoining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 18A-18G. The plasmid vectoris derived from an approximately 2975 base pair SmaI to HindIIIrestriction fragment of psP65 (Promega). Fragment 1 is an approximately1704 base pair SmaI to SmaI restriction sub-fragment of the IBR HindIIIrestriction fragment K (7). Fragment 2 is an approximately 413 base pairSalI to BamHI restriction fragment #7 (3). Fragment 5 is anapproximately 742 base pair NheI to BglI sub-fragment of an IBR virus2400 base pair SmaI fragment. This SmaI fragment has been cloned intothe SmaI site of the plasmid pSP64 (Promega). This plasmid is designatedPSY1645. PSY1645 was deposited on Jul. 16, 1991 with the American TypeCulture Collection. Note that the lacZ marker gene is flanked by XbaIsites located at Junction B and Junction E in this plasmid permittingthe marker gene to be cut out with XbaI.

Homology Vector 591-21.20

The plasmid 591-21.20 was constructed for the purpose of deleting aportion of the IBR thymidine kinase gene. It may also be used to insertforeign DNA into IBR. It contains a unique BglII restriction enzyme siteinto which foreign DNA may be inserted. It may be constructed utilizingstandard recombinant DNA techniques (6, 14) by joining restrictionfragments from the following sources with the synthetic DNA sequencesindicated in FIG. 24. The plasmid vector is derived from anapproximately 2999 base pair SalI to SalI restriction fragment of pSP64(Promega). Fragment 1 is an approximately 1400 base pair SalI to NarIrestriction subfragment contained on the approximately 2700 base pairSalI-SalI restriction subfragment of the IBR HindIII restrictionfragment A (72). Fragment 2 is an approximately 1215 base pair BglIII toSalI restriction subfragment contained on the approximately 2700 basepair SalI-SalI restriction subfragment of the IBR HindIII restrictionfragment A (72).

Homology Vector 552-46.12

The plasmid 591-46.12 was constructed for the purpose of deleting aportion of the Tk gene coding region from the IBR virus. It incorporatesan E. coli β-glucuronidase (uidA) marker gene flanked by IBR virus DNA.The uidA marker gene was inserted into the homology vector 591-21.20 atthe unique BglII site. The marker gene is oriented in the same directionas the Tk gene in the homology vector. A detailed description of themarker gene is given in FIGS. 25A-25B. It may be constructed utilizingstandard recombinant DNA techniques (6, 14) by joining restrictionfragments from the following sources with the synthetic DNA sequencesindicated in FIGS. 25A-25B. Fragment 1 is an approximately 404 base pairSalI to EcoRI restriction subfragment of the PRV BamHI restrictionfragment #10 (3). Note that the EcoRI site was introduced at thelocation indicated in FIGS. 12A-12H by PCR cloning. Fragment 2 is anapproximately 1823 base pair EcoRI to SmaI fragment of the plasmidpRAJ260 (Clonetech). Note that the EcoRI and SmaI sites were introducedat the locations indicated in FIGS. 25A-25B by PCR cloning. Fragment 3is an approximately 784 base pair SmaI to SmaI restriction subfragmentof the HSV-1 BamHI restriction fragment Q (10). Note that this fragmentis oriented such that the polyadenylation sequence (AATAAA) is locatedclosest to junction C.

Homology Vector 691-096.2

The homology vector 691-096.2 was constructed for the purpose ofinserting foreign DNA into the unique long of IBRV. It was constructedutilizing standard recombinant DNA techniques (6), by joiningrestriction fragments from the following sources. The plasmid vector wasderived from an approximately 2485 base pair Nael to PvuII restrictionfragment of pSP65 (Promega). Fragment 1 is an approximately 3900 basepair ApaI to ApaI restriction sub-fragment within the larger BamHI-KpnIsubfragment of the IBRV BamHI restriction fragment C (7). Foreign DNA isinserted into a unique HindIII or XbaI restriction endonuclease sitewithin the ApaI to ApaI restriction subfragment.

Homology Vector 756-11.17

The homology vector 756-11.17 was constructed for the purpose ofinserting foreign DNA into the gG deletion within the unique shortregion of IBRV and was derived from homology vector 439-70.4. Itincorporates an E. coli uidA marker gene and PI-3 HN (amino acids 1-573)and F genes (amino acids 4-450) flanked by IBRV DNA. The first IBRVregion is an approximately 888 base pair fragment of IBR virus DNA whichbegins approximately 1042 base pairs upstream of the initiation codon ofthe gG gene and ends approximately 154 base pairs upstream of theinitiation codon of the gG gene. The second IBRV region is anapproximately 785 base pair fragment of IBR virus DNA which begins withsequences encoding the last 80 amino acids of the gG primary translationproduct. Inserted into the IBRV gG deletion region between the first andsecond regions is a synthetic polylinker adding restriction endonucleasesites for SwaI-BglII-SwaI-HindIII-BamHI-SpeI. The lacZ gene was insertedinto the SwaI site of the polylinker, and the PI-3 F and HN genes wereinserted into the HindIII site of the polylinker. The PI-3 F and HNgenes were isolated by CLONING OF PARAINFLUENZA-3 FUSION ANDHEMAGGLUTININ GENES. The homology vector 756-11.17 was constructedutilizing standard recombinant DNA techniques (6), by joiningrestriction fragments from the following sources. The plasmid vector wasderived from an approximately 2965 base pair HindIII to SmaI restrictionfragment of pSP64 (Promega) Fragment 1 is an approximately 3593 basepair HindIII to XhoI restriction fragment of the IBRV HindIIIrestriction fragment K (7). Fragment 2 is a HSV-1 TK promoter lacZ geneDNA fragment inserted into the SwaI site of the synthetic polylinker.Fragment 3 is a HCMV IE promoter PI-3 F gene/PRV gX promoter PI-3 HNgene DNA fragment inserted into the HindIII site of the syntheticpolylinker. Fragment 4 is an approximately 785 base pair XhoI to NdeIrestriction fragment of the IBRV HindIII restriction fragment K (7).

Homology Vector 769-73.1

The homology vector 769-73.1 was constructed for the purpose ofinserting foreign DNA into the repeat region of IBRV. It was constructedutilizing standard recombinant DNA techniques (6), by joiningrestriction fragments from the following sources. The plasmid vector wasderived from an approximately 2792 base pair SmaI to PvuII restrictionfragment of pSP65 (Promega). Fragment 1 is an approximately 2900 basepair NruI to XhoI restriction sub-fragment within the smaller KpnI-BamHIsubfragment of the IBRV BamHI restriction fragment C (7). A syntheticpolylinker was inserted into a unique EcoRV site within the 2900 basepair Nrul to XhoI fragment. The synthetic polylinker provides uniquerestriction sites: SwaI-BglII-SwaI-HindIII-BamHI-SpeI.

EXAMPLES Example 1

S-IBR-002

S-IBR-002 is an IBR virus that has a deletion of approximately 800 bp inthe repeat region of the genome. This deletion removes the only twoEcoRV restriction sites on the virus genome and an adjacent BglII site(FIG. 2).

To construct this virus, the DIRECT LIGATION PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS was performed. Purified IBR virus DNA (Cooperstrain) digested with EcoRV restriction enzyme was mixed withDraI-restriction enzyme-digested plasmid DNA containing the E.coliβ-galactosidase (lacZ) gene under the control of the HSV-1 TK promoter.After ligation the mixture was used to transfect animal cells and thetransfection stock was screened for recombinant IBR virus by theSOUTHERN BLOTTING OF DNA procedure. The final result of the purificationwas the recombinant IBR virus designated S-IBR-002. It was shown bySouthern hybridization that this virus does not carry any foreign genes.Restriction enzyme analysis also showed that the insertion sites (EcoRV)in both repeats were deleted. FIG. 2 shows the restriction map of theEcoRI B fragment which contains the EcoRV restriction sites and the mapof S-IBR-002 which lacks the EcoRV sites. S-IBR-002 was deposited onJun. 18, 1986 pursuant to the Budapest Treaty on the InternationalDeposit of Microorganisms for the Purposes of Patent Procedure with thePatent Culture Depository of the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR2140.

A study was conducted to determine the safety and serological responseof young calves following intramuscular administration of S-IBR-002.These results are presented in Table 1. Three calves were inoculatedintramuscularly with 10⁷ PFU of S-IBR-002. Clinical signs of IBR andfebrile response were absent in these calves, as well as in the contactcontrol calf. All three calves developed significant neutralizingantibody to IBR virus but the contact control remained seronegative.These results suggest that S-IBR-002 is useful as a vaccine against IBRdisease.

TABLE 1 Serologic and Clinical Response of Young Calves FollowingVaccination with S-IBR-002 Clinical and Antibody Titer Virus CalfFebrile Virus Days Post Inoculation Construct # response Isolation^(a) 07 14 21 28 S-IBR-002 28 NONE (—) <2 <4 6  5 3 30 NONE (—) <2 <4 6 <2 694 NONE (—) <2 <4 6  3 8 Control 32 NONE (—) <4 <4 <4  <2 <4  ^(a)Fromnasal swabs and peripheral blood leukocytes.

Example 2

Unique Short 2 Gene

The unique short region of IBR virus contains a gene homologous to theUS2 gene of several other herpesviruses. In the studies described belowdeletion of the IBR unique short 2 gene (US2) may render the virus safefor use in pregnant cows, as determined by direct fetal inoculation.

Observing that the Nasalgen IBR vaccine strain will not cause abortionwhen used in IBR-susceptible pregnant cows at various stages ofgestation (18,65), the genomic lesion responsible for this property aredetermined. The genome of this virus are characterized by restrictionmapping and DNA sequence analysis. It was determined that a majorportion of the IBR virus US2 gene was deleted from the Nasalgen virus.Restriction mapping of the Nasalgen virus indicated that the HindIII Kfragment contained an approximately 800 base pair deletion. The deletionwas localized to the end of the HindIII K Fragment located next to theHindIII O fragment (see FIG. 1). Therefore, the HindIII K fragment fromthe Cooper strain was subcloned and this region was sequenced. The first1080 base pairs of the fragment were found to contain an open readingframe (ORF) coding for 309 amino acids (see FIG. 3). The ORF is 68% G+Cand encodes a protein with a predicted molecular weight of 46,094.Comparison of the sequence of the predicted protein with sequences ofgene products of HSV-1, PRV, HSV-2, and marek's disease virus in theunique short region indicated that this ORF is homologous to theherpesvirus US2 gene (see FIGS. 4A-4B). Although the function of theherpesvirus US2 gene is not known, the gene has been shown to benonessential for growth of HSV in cell culture (4,19). The US2 gene hasalso been shown to be deleted in the PRV vaccine strains Norden andBartha (11).

The HindIII K fragment from the Nasalgen virus was subcloned and thedeletion region was sequenced. When the sequence obtained from theNasalgen strain was compared to the sequence obtained from the Cooperstrain (see FIGS. 5A-5B), it was possible to determine that amino acids59 to 309 of the US2 gene had been deleted. It was also determined thatmost of the HindIII O fragment had also been deleted.

Cattle studies have shown that the Nasalgen virus will not causeabortion when used in IBR-susceptible pregnant cows at various stages ofgestation (18). Since the only major difference between the wild-typeIBR strain and the Nasalgen strain resides in the deletion of the US2gene, this gene may be involved in the fetal virulence observed for thewild type virus.

Example 3

S-IBR-027

S-IBR-027 is an IBR virus that has a deletion of approximately 800 bp inthe repeat regions and approximately 1200 bp in the short unique regionof the genome. The deletion in the short unique region removes the US2gene (FIG. 6). The repeat deletion was derived from the parental virusS-IBR-002 and is described in Example 2.

To construct this virus, the HOMOLOGOUS RECOMBINATION PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS was performed. A homology vectorcontaining the bacterial transposon Tn5 NEO (aminoglycosidase3′-phosphotransferase) gene under the control of the HSV α4 promoterflanked by sequences from the IBR virus TK gene was constructed. The IBRvirus homology regions were derived from the TK gene. The upstreamhomology included the first amino acid of the TK gene (15) and extendedapproximately 800 base pairs upstream of the TK coding region. Thedownstream homology included amino acids 156 to 357 and extendeddownstream of the TK coding region approximately 60 base pairs.S-IBR-002 DNA was mixed with the homology vector and transfected intorabbit skin cells as indicated in the methods. The transfection stockwas selected according to the SELECTION OF G418 RESISTANT IBR VIRUS.Individual clones were picked after one round of selection and analyzedby the SOUTHERN BLOTTING OF DNA procedure. When a probe derived from theNEO gene was used in this analysis, one clone was found which did nothybridize to the NEO probe but had a HindIII restriction digestionpattern clearly distinct from the parental S-IBR-002. Further analysisindicated that the NEO had not been inserted into the TK region, howeveran approximately 1200 base pair deletion had occurred in the HindIII Kfragment.

In order to characterize the HindIII K deletion, that fragment wassubcloned and subjected to restriction mapping. Utilizing a series ofoligonucleotide probes derived from the wild type sequence it wasdetermined that approximately 1200 base pairs were deleted from the endof the HindIII K fragment adjacent to the HindIII K/HindIII O junction(see FIG. 6). This deletion removes the entire coding region of the US2gene. S-IBR-027 was deposited on Apr. 17, 1991 pursuant to the BudapestTreaty on the International Deposit of Microorganisms for the Purposesof Patent Procedure with the Patent Culture Depository of the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852U.S.A. under ATCC Accession No. VR 2322.

Direct fetal inoculation is the most sensitive test for determining thesafety of live, IBR vaccines as regards their use in pregnant cows or incalves nursing pregnant cows. Three virus constructs were tested forfetal safety by inoculating directly into the bovine fetus, followinglaparotomy to expose the uterus. Abortion occurring within seven daysafter inoculation was considered to be surgically-induced. If fetusesaborted after this time, tissue samples were removed and cultured forthe presence of the IBR construct. Caesarean sections were performed oncows with fetuses surviving for greater than 30 days post-inoculation.Fetal tissue was removed for virus culturing and blood samples weretaken for evaluation of serum antibody to IBR virus.

The S-IBR-027 construct described above was tested, as well as two otherconstructs, S-IBR-020 and S-IBR-028. The S-IBR-020 construct was derivedfrom the Cooper strain of IBR virus by making deletions in the repeatregions of the DNA and by inserting the Tn5 NEO gene. The S-IBR-028construct was derived from the Cooper strain of IBR virus by makingdeletions in the repeat region of the DNA and in the TK gene. The Tn5NEO gene was also inserted into the TK deletion.

The following results were obtained from studies with the three virusconstructs. In the studies with S-IBR-020, two fetuses were inoculated,one at approximately 130-140 days gestation and the other atapproximately 170-180 days gestation. The younger fetus aborted twentydays after inoculation, but virus could not be recovered from tissuesamples of this fetus (Table 2). The other fetus was live and appearednormal when it was surgically removed 60 days post-inoculation. Instudies with S-IBR-027, four fetuses, ranging in age from 125 daysto >250 days, were inoculated (Table 2). All fetuses survived andappeared normal. In studies with S-IBR-028, three fetuses, ranging inage from 140 days to >250 days, were inoculated. The youngest and eldestfetuses survived and appeared normal, however the fetus inoculated at160-170 days gestation aborted nine days after inoculation.

Direct fetal inoculation is the most sensitive test for measuring thesafety of live, IBR viruses used in pregnant cows. To date, the gene(s)involved in fetal virulence has not been reported. IBR viruses wasengineered with deletions in three different regions of IBR virus DNAand then determined the effect of the gene deletion. All three virusconstructs tested have a deletion in the repeat region of the DNA andtwo constructs do not have TK activity. One fetus inoculated with eachof the TK-constructs has aborted. In contrast, the construct withdeletions in the repeat regions and the US2 gene (S-IBR-027) has beeninoculated into four fetuses with no adverse reactions.

TABLE 2 Safety of IBR Viruses for Bovine Fetuses Construct Fetal Age^(a)Results S-IBR-020 130-140 Days Fetus aborted Day 20 post- inoculation;no virus isolated 170-180 Days Normal, live fetus 60 days post-inoculation S-IBR-027 125-135 Days Normal, live fetus 60 days post-inoculation 150-160 Days Normal, live calf born 56 days post-inoculation220-240 Days Normal, live calf born 30 days post-inoculation >250 DaysNormal, live calf born 30 days post-inoculation S-IBR-028 140-150 DaysNormal, live fetus 60 days post- inoculation 160-170 Days Fetus abortedDay 9 post- inoculation; no virus isolated >250 Days Normal, live calfborn 12 days post-inoculation ^(a)Approximate age at time of virusinoculation

S-IBR-027 is safe for fetal inoculation in contrast to S-IBR-020 andS-IBR-028 which are not. Although all three viruses were engineered bysimilar approaches, the distinguishing difference of S-IBR-027 is thedeletion of the US2 gene. Nasalgen virus, which was generated byindependent methods, is also safe for use in IBR-susceptible pregnantcows, has been deleted in the US2 gene.

Although the S-IBR-027 and Nasalgen have the similar property of fetalsafety, S-IBR-027 offers additional advantages. The major portion of theUS2 gene (251 out of 309 amino acids) has been deleted in the Nasalgenvirus. This deletion would clearly inactivate the gene, however theremaining portion of the gene may make it more likely to revert tovirulence via recombination with other viruses. The complete codingregion of the US2 has been deleted from S-IBR-027 making it less likelythat this gene could be restored and revert the virus to virulence. TheS-IBR-027 construct also carries an important deletion in the repeatregion, which is not present in the Nasalgen virus. A deletion in theanalogous region of the pseudorabies virus (PRV) has been shown to bevaluable in attenuating PRV for swine (see U.S. Pat. No. 4,877,737).This deletion has also been shown to attenuate IBR for cattle as seen inthe testing of S-IBR-002 (see Example 1).

Example 4

S-IBR-028

S-IBR-028 is an IBR virus that has a deletion of approximately 800 bp inthe repeat regions and approximately 250 bp in the TK region of thegenome. The deletion in the TK region inactivates the TK gene. Therepeat deletion was derived from the parental virus S-IBR-002 and isdescribed in Example 2.

To construct this virus, the HOMOLOGOUS RECOMBINATION PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS was performed. A homology vectorcontaining the bacterial transposon Tn5 NEO (aminoglycosidase3-phosphotransferase) gene under the control of the HSV-1 α4 genepromoter flanked by sequences from the IBR virus TK gene wasconstructed. The IBR virus homology regions were derived from the TKgene. The upstream homology included amino acids 1 to 62 of the TK gene(15) and extended approximately 674 base pairs upstream of the TK codingregion. The downstream homology included amino acids 156 to 357 andextended downstream of the TK coding region approximately 1138 basepairs. S-IBR-002 DNA was mixed with the homology vector 129-71.5 andtransfected into rabbit skin cells as indicated in the methods. Thetransfection stock was selected according to the SELECTION OF G418RESISTANT IBR VIRUS.

Individual clones were picked after two rounds of selection and analyzedby the SOUTHERN BLOTTING OF DNA procedure. Several clones were assayedfor TK activity by a ¹⁴C-thymidine incorporation assay (29). One clonewhich was negative for TK activity was chosen and characterized bydigestion with HindIII and XbaI. The restriction endonuclease analysisconfirmed that the NEO gene had been inserted into the TK gene. Thisclone, designated S-IBR-028, was deposited on May 14, 1991 pursuant tothe Budapest Treaty on the International Deposit of Microorganisms forthe Purposes of Patent Procedure with the Patent Culture Depository ofthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. 20852 U.S.A. under ATCC Accession No. VR 2326.

Example 5

Glycoprotein G Gene

Deletion of the PRV gX gene has been shown to be valuable both as anattenuating lesion and as a negative serological marker (see U.S. Ser.No. 192,866, filed May 11, 1988 now U.S. Pat. No. 5,047,237 issued Sep.10, 1991). In the studies described below the unique short region of IBRvirus was shown to contain a gene homologous to the gX gene of PRV.

The sequence of an approximately 1400 base pair region of the IBRHindIII K fragment (see FIG. 8), located approximately 2800 base pairsdownstream of the HindIII K/HindIII O junction was determined. Thisregion was found to contain an ORF coding for 441 amino acids translatedin the direction away from the HindIII K/HindIII O junction (see FIG.1). The ORF is 69% G+C and encodes a protein with a predicted molecularweight of 58,683. Comparison of the sequence of the predicted proteinwith sequences of gene products of HSV-2 and PRV in the unique shortregion indicated that this ORF is homologous to the herpesvirus gG gene(see FIGS. 9A-9B). The complete gG gene resides on an approximately 2800base pair MluI to NdeI sub-fragment of the IBR virus HindIII K fragment.This subfragment has been cloned as a blunt ended fragment into theplasmid pSP64. This plasmid is designated PSY1643. PSY1643 was depositedon Jul. 16, 1991 pursuant to the Budapest Treaty on the InternationalDeposit of Microorganisms for the Purposes of Patent Procedure with thePatent Culture Depository of the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No.68652. This plasmid may be used to confirm the sequence of the gG gene.The sequence of the gG gene may also be confirmed by comparing theappropriate DNA sequence of the wild type virus S-IBR-000 (Cooper strainwith the sequence of the gG deleted virus S-IBR-037 (ATCC Accession No.2320).

To confirm the expression of the IBR virus gG gene product, cells wereinfected with IBR virus and samples of media from infected cultures weresubjected to SDS-polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. The anti-serum usedwas a mouse hyper-immune serum raised against chemically-synthesized gGpeptides (amino acids 242-254 and 269-289) linked to keyhole limpethemocyanin. As shown in FIG. 10, gG is prominent in the media of cellsinfected with wild type virus (S-IBR-000), but is not detected in mediaof mock infected cells.

Example 6

S-PRV-160

S-PRV-160 is a pseudorabies virus that has a deletion in the TK gene inthe long unique region, a deletion in the repeat region, and anapproximately 1414 base pair deletion in the gX coding region. The genefor E.coli β-galactosidase (lacZ gene) was inserted in the place of thegX gene and is under the control of the gX promoter. A chimeric genecoding for an IBR virus gG, PRV gIII and PRV gX fusion protein wasinserted at the HindIII sites located in each repeat.

S-PRV-160 was constructed utilizing plasmid 459-12.6, pseudorabies virusS-PRV-013 (see U.S. Ser. No. 823,102, filed Jan. 27, 1986 now U.S. Pat.No. 5,068,192 issued Nov. 26, 1991 and U.S. Ser. No. 07/192,866, filedMay 11, 1988 now U.S. Pat. No. 5,047,237 issued Sep. 10, 1991) and therestriction enzyme HindIII in the DIRECT LIGATION PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS. Several clones were screened bydigestion with HindIII for the presence of the HindIII band containingthe chimeric gene insert from plasmid 459-12.6. One clone exhibiting thecorrect HindIII insert band was chosen and designated S-PRV-160.

S-PRV-160 was constructed so that it would express precisely the gGspecific amino acids that were deleted in S-IBR-037. This allows the gGfusion protein expressed in S-PRV-160 to be used as an antigen toidentify antibodies directed against the wild type virus as opposed toantibodies directed against S-IBR-037. Note that gX, the PRV homologueof IBR virus gG, has been deleted from S-PRV-160 to prevent anyconfusion resulting from cross reactivity that might exist between thetwo proteins. To confirm that S-PRV-160 does express IBR virus gG, aWestern blot analysis was performed. As can be seen in FIG. 10, gGspecific antibody does react with an appropriately sized media proteinfrom S-PRV-160.

S-PRV-160 may also be utilized as an antigen for the production of gGspecific monoclonal antibodies. Such antibodies are useful in thedevelopment of diagnostic tests specific for the gG protein. Monoclonalantibodies were generated in mice utilizing S-PRV-160 according to thePROCEDURE FOR GENERATING MONOCLONAL ANTIBODIES. One of these antibodies'clone 3-1G7 was shown to react specifically with purified gG in the gGELISA assay.

Example 7

S-IBR-035

S-IBR-035 is an IBR virus that has two deletions in the short uniqueregion of the genome. The first deletion is approximately 2500 basepairs and begins in the HindIII K fragment approximately 1750 base pairsdownstream of the HindIII O/HindIII K junction and extends back throughthat junction. This deletion removes the US2 gene. The second deletionis approximately 294 base pairs and begins in the HindIII K fragmentapproximately 3900 base pairs downstream of the HindIII K/HindIII Ojunction and extends back toward that junction. This deletion removesamino acids 263 to 361 of the gG gene. The gene for E.coliβ-galactosidase (lacZ gene) was inserted into the deletion in the gGgene and is under the control of the HCMV immediate early promoter.

S-IBR-035 was derived from S-IBR-000 (Cooper strain). This wasaccomplished utilizing the homology vector 439-01.31 (see Materials andMethods) and virus S-IBR-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock wasscreened by the BLUOGAL™ SCREEN FOR RECOMBINANT HERPESVIRUS. The finalresult of blue plaque purification was the recombinant virus designatedS-IBR-035. This virus was characterized by restriction mapping and theSOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertionof the β-galactosidase (lacZ) marker gene and the deletion ofapproximately 294 base pairs of the gG gene. It was also confirmed thatan approximately 2500 base pair deletion had occurred in the region ofthe US2 gene.

Example 8

S-IBR-036

S-IBR-036 is an IBR virus that has two deletions in the short uniqueregion of the genome. The first deletion is approximately 2500 basepairs and is similar to the deletion in S-IBR-035 (see Example 7) whichremoves the US2 gene. The second deletion is approximately 1230 basepairs and begins in the HindIII K fragment approximately 3900 base pairsdownstream of the HindIII O/HindIII K junction and extends back towardthat junction. This deletion removes amino acids 1 to 361 of the gGgene. The gene for E.coli β-galactosidase (lacZ gene) was inserted intothe deletion in the gG gene and is under the control of the HCMVimmediate early promoter.

S-IBR-036 was derived from S-IBR-000 (Cooper strain). This wasaccomplished utilizing the homology vector 439-21.69 (see Materials andMethods) and virus S-IBR-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock wasscreened by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. The finalresult of blue plaque purification was the recombinant virus designatedS-IBR-036. This virus was characterized by restriction mapping and theSOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertionof the β-galactosidase (lacZ) marker gene and the deletion ofapproximately 1230 base pairs of the gG gene. It was also confirmed thatan approximately 2500 base pair deletion had occurred in the region ofthe US2 gene (see above).

Example 9

S-IBR-037

S-IBR-037 is an IBR virus that has two deletions in the short uniqueregion of the genome. The first deletion is approximately 2500 basepairs and begins in the HindIII K fragment approximately 1750 base pairsdownstream of the HindIII O/HindIII K junction and extends back throughthat junction. This deletion removes the US2 gene. The second deletionis approximately 1230 base pairs and begins in the HindIII K fragmentapproximately 3900 base pairs downstream of the HindIII O/HindIII Kjunction and extends back toward that junction. This deletion removesamino acids 1 to 361 of the gG gene.

S-IBR-037 was derived from S-IBR-035. This was accomplished utilizingthe homology vector 439-70.4 (see Materials and Methods) and virusS-IBR-035 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS. The transfection stock was screened by theBLUOGAL™ SCREEN FOR RECOMBINANT HERPESVIRUS. The result of white plaquepurification was the recombinant virus designated S-IBR-037. This viruswas characterized by restriction mapping and the SOUTHERN BLOTTING DNAprocedure. This analysis confirmed the deletion of the β-galactosidase(lacZ) marker gene and the deletion of approximately 1230 base pairs ofthe gG gene. It was also confirmed that an approximately 2500 base pairdeletion had occurred in the region of the US2 gene (see above).S-IBR-037 was deposited on Apr. 16, 1991 pursuant to the Budapest Treatyon the International Deposit of Microorganisms for the Purposes ofPatent Procedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. VR 2320.

To test the efficacy of S-IBR-037 as an inactivated IBR virus vaccine inprotecting susceptible calves against virulent IBR virus challenge, astudy was performed according to the VACCINATION STUDIES IN CALVES WITHINACTIVATED IBR VIRUS. The following results were observed.

Virus neutralization antibody titers were elicited in animals after thefirst vaccination (see Table 3). Antibody titers were not significantlydifferent between animals that received a vaccine dose of 10^(7.3) virusand animals vaccinated with 10^(8.0) virus. After the secondvaccination, mean antibody titers increased to 1:19 and 1:32,respectively, for the 10^(7.3) and 10^(8.0) vaccine groups. Controlanimals remained seronegative to IBR virus throughout the vaccinationperiod. Antibody titers in both vaccinate groups showed an increasetypical of an anamnestic response after challenge with virulent IBRvirus. By 13 days post challenge, mean antibody titers were 1:152 and1:215 for the 10^(7.3) and 10^(8.0) vaccinate groups respectively. Incontrast, mean antibody titers in challenged control animals were 1:4 at7 days and 1:8 at 13 days post challenge.

Nasal swabs were collected from challenged animals to determine whethervaccination decreased the time of virus shedding (Table 4). The mostdramatic difference between vaccinates and control animals was observedat 12 days post challenge. At this time, seventy-five percent of controlanimals continue to shed, whereas, only twenty-five percent of bothvaccinate groups shed virus. Virus was not isolated from control orvaccinated groups at 15 days post challenge.

TABLE 3 Generation of virus neutralizing antibody in animals vaccinatedwith inactivated S-IBR-037 vaccine. Antibody titer^(a) on days: PostPost Vaccination Challenge Animal No. 7 21 28 42 7 13 Controls  9 ≦2 ≦2≦2 ≦2 4 4 22 ≦2 ≦2 ≦2 ≦2 4 8 32 ≦2 ≦2 ≦2 ≦2 4 16 64 ≦2 ≦2 ≦2 ≦2 4 8 GMT≦2 ≦2 ≦2 ≦2 4 8 Vaccinates dose 10^(7.3)  1 ≦2 8 32 64 64 128 20 ≦2 8 3264 64 256 25 ≦2 8 16 8 64 512 36 ≦2 4 16 4 16 ≧32 GMT ≦2 6.7 22.6* 19.0*45.34 152.2 Vaccinates dose 10^(8.0)  7 ≦2 4 32 8 64 256 30 ≦2 ≧8 64 128128 ≧128 33 ≦2 16 32 128 128 256 69 ≦2 4 16 8 128 256 GMT ≦2 6.7 32* 32*107.6 215.3 *Statistically greater than controls (p ≦ 0.05)^(a)Expressed as reciprocal of dilution.

TABLE 4 Isolation of IBR virus from vaccinated and unvaccinated controlanimals after challenge with virulent IBR virus. IBR virus isolated(+/−) from animals on days post challenge Animal No. 3 6 9 12 15Controls  9 − + + + − 22 − + + − − 32 − + + + − 64 − + + + − Vaccinatesdose 10^(7.3)  1 − + + − − 20 − + + − − 25 − + + − − 36 − + + + −Vaccinates dose 10^(8.0)  7 − + + − − 30 − − − − − 33 − + + + − 69 − + +− −

TABLE 5 Vaccinated animals demonstrate reduced clinical signs of IBR.Clinical scores post challenge Animal Serious Mucopurulent Tem- No.Attitude^(a) Ulcers^(b) Discharge^(c) Discharge^(d) perature^(e)Controls  9 5 3 11 5 3 22 2 2 12 3 1 32 5 3 11 0 4 64 6 3 11 1 1 GMS 4.52.8 11.3 2.3 2.3 Vaccinates dose 10^(7.3)  1 0 2 1 0 0 20 0 1 3 0 0 25 02 6 2 0  36^(f) 6 2 1 13 0 GMS 1.5 1.8 2.8* 2.3 0 Vaccinates dose10^(8.0)  7 1 2 1 0 0 30 1 2 2 2 0 33 1 2 0 0 0 69 1 2 0 0 0 GMS 1 20.8* 0.5 0 ^(a)Days with depressed attitude. ^(b)Number of ulcers.^(c)Days with serous discharge. ^(d)Days with mucopurulent discharge.^(e)Days with ≧2° F. above baseline temperature. ^(f)Animal exhibitedmucopurulent discharge on the day of challenge and for 13 days postchallenge. *Statistically greater than controls (p ≦ 0.05)

Animals were observed daily for 13 days post challenge for clinicalsigns of IBR infection. Clinical disease was evaluated with respect toattitude, the number of ulcers, extent of serious and mucopurulentdischarge and the number of days with elevated temperature. The resultspresented in Table 5 show that vaccinated animals exhibited less severedisease than did unvaccinated control animals. Control animals showedclinical depression (“Attitude” in Table 5) for 4.5 days compared with 1to 1.5 days for vaccinated animals. The amount and extent of serousdischarge was substantially reduced in both vaccinate groups comparedwith controls. The extent of mucopurulent discharge was also reduced invaccinated animals, although to a lesser degree. However, vaccinateanimal #36 did have mucopurulent discharge on the day of challenge andis not consistent with the results for other vaccinates. None of thevaccinates exhibited temperatures of ≧2° F. above baseline. In contrast,all control animals exhibited elevated temperatures of ≧2° F. overbaseline and 2 of 4 control animals had temperatures of 104° F. andabove.

Vaccination of calves with inactivated S-IBR-037 vaccine protected theanimals against virulent wild-type IBR virus challenge. Virusneutralization titers were statistically greater in vaccinated than incontrol animals. An anamnestic response in antibody titer was observed 7days post challenge, indicating the development of humoral memoryresponse. Except for 7 days post challenge, neutralization titersbetween the 10^(7.3) and 10^(8.0) vaccinate groups were notstatistically different. Fewer vaccinated animals shed virulentchallenge virus than control animals. These results suggest thatvirulent IBR virus is cleared more rapidly in vaccinated than inunvaccinated animals. Clinical symptoms of IBR virus infection were alsoreduced in vaccinated animals. After challenge, both vaccinate groupsexhibited fewer days of depressed attitude, reduced serous discharge,and no elevated temperature compared with controls.

In order to show that gG antibody is produced in vaccinated calvesfollowing exposure to wild-type virus, serum samples taken pre- andpost-exposure to wild-type viruses were subjected to the ELISA assay.Samples taken at the day of challenge and at 13 days post-challenge wereanalyzed. As seen in Table 6, the post-challenge absorbance readings forgG increase for each animal (ratio of >1.0), indicating that within 13days of infection a detectable immune response to gG is present.

TABLE 6 Detection of antibody to gG in serum of animals vaccinated withS-IBR-037 and challenged with wild type. Animal No. Ratio of pre- vs.post challenge^(a) Controls  9 1.22 22 1.96 32 1.87 64 2.19 Vaccinatesdose 10^(7.3)  1 1.39 20 1.40 25 1.84 36 1.18 Vaccinates dose 10^(8.0) 7 1.19 30 1.29 33 1.52 69 2.66 ^(a)Animals were challenged with10^(7.6) PFU of wild type IBR virus. Pre-challenge serum from day ofchallenge, post-challenge serum from 13 days post challenge. Datareflects the average of the ratio of absorbance readings for threeindependent ELISA determinations.

Example 10

S-IBR-038

S-IBR-038 is an IBR virus that has two deletions in the short uniqueregion of the genome. The first deletion is approximately 2500 basepairs and begins in the HindIII K fragment approximately 1750 base pairsdownstream of the HindIII O/HindIII K junction and extends back throughthat junction. This deletion removes the US2 gene. The second deletionis approximately 294 base pairs and begins in the HindIII K fragmentapproximately 3900 base pairs downstream of the HindIII K/HindIII Ojunction and extends back toward that junction. This deletion removesamino acids 261 to 359 of the gG gene.

S-IBR-038 resulted from the removal of the marker gene from S-IBR-035(see above). This was accomplished by digestion of S-IBR-035 with XbaIas described in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANTHERPESVIRUS. The structure of S-IBR-035 was confirmed by restrictionenzyme analysis with HindIII, BamHI and XbaI.

Example 11

Glycoprotein E Gene

Deletion of the PRV gI gene has been shown to be valuable both as anattenuating lesion and a negative serological marker (3,42). In thestudies described below the unique short region of infectious bronchitisvirus virus was shown to contain a gene homologous to the gI gene ofPRV.

The sequence of 2038 base pairs of the IBR unique short region, startingapproximately 1325 base pairs upstream of the HindIII K/HindIII Fjunction in the HindIII K fragment was determined. This region was foundto contain an ORF coding for 617 amino acids translated in the directionaway from the HindIII K/HindIII O junction (see FIG. 1). The ORF is70.5% G+C and encodes a protein with a predicted molecular weight ofapproximately 88,980. Comparison of the sequence of the predictedprotein with sequences of gene products of HSV-1, VZV, and PRV in theunique short region indicated that this ORF is homologous to theherpesvirus gE gene (see FIGS. 16A-16B).

The DNA encoding the gE gene has been cloned in two plasmids, PSY1644and PSY1645. The amino-terminal half of the gene (encoding amino acids1-276) was cloned as an approximately 2300 base pair fragment resultingfrom a partial SmaI digest of wild type S-IBR-000 (Cooper Strain) DNA.This fragment was inserted into the plasmid pSP64 to yield PSY1644. Thisplasmid, designated PSY1644, was deposited on Jul. 16, 1991 pursuant tothe Budapest Treaty on the International Deposit of Microorganisms forthe Purposes of Patent Procedure with the Patent Culture Depository ofthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. 20852 U.S.A. under ATCC Accession No. 68651. The carboxyl-terminalhalf of the gene (encoding amino acids 277-617) was cloned as anapproximately 2400 base pair SmaI fragment. The fragment was insertedinto the plasmid pSP64 to yield PSY1645. This plasmid, designatedPSY1645, was deposited on Jul. 16, 1991 pursuant to the Budapest Treatyon the International Deposit of Microorganisms for the Purposes ofPatent Procedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. 68650. These plasmids may be used to confirmthe sequence of the gE gene.

Example 12

Pseudorabies Virus Expressing IBR Virus qE

A pseudorabies virus analogous to S-PRV-160 may be constructed for thepurpose of expressing the IBR virus gE. This may be accomplished byinserting the gene coding for IBR virus gE into S-PRV-002 (U.S. Pat. No.4,877,737).

Such an expression vector may be constructed utilizing the IBR virus gEplasmid described in the methods section, pseudorabies virus S-PRV-002and the restriction enzyme XbaI in the DIRECT LIGATION PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS. Viruses resulting from thisprocedure may be screened by digestion with XbaI for the presence of theXbaI band containing the IBR virus gE gene.

The gE protein expressed from this vector may be used as an antigen toidentify antibodies directed against the wild type virus as opposed toantibodies directed against gE deleted viruses. This virus may also beutilized as an antigen for the production of gE specific monoclonalantibodies. Such antibodies are useful in the development of diagnostictests specific for the gE protein. Monoclonal antibodies may begenerated in mice utilizing this virus according to the PROCEDURE FORGENERATING MONOCLONAL ANTIBODIES.

Example 13

Glycoprotein E Deleted IBR Viruses

The HOMOLOGY VECTOR 536-03.5 was used to generate various gE-deleted IBRviruses. Utilizing the general strategy described in CONSTRUCTION OFDELETION VIRUSES, a gE deletion of approximately 1410 base pairs (aminoacids 77-547) was introduced into two different IBR virus backbones,S-IBR-000 (Cooper Strain) and S-IBR-037. The virus resulting from theS-IBR-000 parent contains the gE deletion alone. The virus resultingfrom the S-IBR-037 parent contains the gE deletion in conjunction withthe US2 and gG deletions. The lacZ marker gene may be removed from theseviruses utilizing the procedures outlined in the methods section.

These gE-deleted viruses are of great value as IBR vaccines. Theircombination of different deletions will provide the varying degrees ofattenuation which are required for a superior vaccine. These viruseswill also provide a negative serological marker which may be used todistinguish vaccinated from infected animals. The virus containing bothgG and gE deletions should be of even greater value by having twonegative markers. The availability of two negative markers permits onemarker to be used as a confirmatory test, greatly increasing thereliability of such a diagnostic determination.

Example 14

S-IBR-004

S-IBR-004 is an IBR recombinant virus carrying an inserted foreign gene,Tn5 NEO (aminoglycoside 3′-phosphotransferase) gene, under the controlof the pseudorabies virus (PRV) glycoprotein X promoter.

To construct this virus, the HindIII K DNA fragment from wild type IBRvirus was cloned into the plasmid pSP64 at the HindIII site. Thisplasmid was designated pSY524. A map of the HindIII K fragment is shownin FIG. 19. The DNA from the XhoI site to the HindIII site andcontaining the NdeI site from pSY524 was cloned into plasmid pSP65 andcalled pSY846. The NdeI to EcoRI fragment was removed from pSY846 bydigestion with NdeI and EcoRI restriction enzymes, followed byPOLYMERASE FILL-IN REACTION and LIGATION. The resulting plasmid wascalled pSY862. The plasmid pNEO (P.L. Biochemicals, Inc.) contains theaminoglycoside 3′-phosphotransferase (NEO) gene and confers resistanceto ampicillin and neomycin on E. coli hosts. The coding region of thisgene (BglII-BamHI fragment) was isolated and cloned between the PRV gXpromoter and the HSV-TK poly A sequence in a plasmid called pSY845.

The NEO gene construct in pSY845 was excised with HindIII, made bluntended by the POLYMERASE FILL-IN REACTION, and cloned into the SacI siteof plasmid pSY862. The final product was called pSY868.

Wild type IBR DNA was mixed with pSY868 DNA and the mixture wastransfected into rabbit skin cells to generate recombinant IBR. Therecombinant IBR virus carrying a functional NEO gene was then isolatedand purified according to the SELECTION OF G418 RESISTANT IBR VIRUSmethod.

S-IBR-004 recombinant IBR was shown to express the NEO gene by the factthat cells infected with this virus were resistant to the toxicity ofG418. A detailed map of the plasmid construction is shown in FIG. 19.The structure of S-IBR-004 is also shown in FIG. 19. S-IBR-004 wasdeposited on May 23, 1986 pursuant to the Budapest Treaty on theInternational Deposit of Microorganisms for the Purposes of PatentProcedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. VR 2134.

Example 15

S-IBR-008

S-IBR-008 is an IBR virus that has a deletion in the short uniqueregion, and an insertion of the bovine rotavirus glycoprotein 38 (g38)gene in the XbaI site in the long unique region. The Xba I site islocated in the intergenic region upstream of the lactency-relatedtranscripts promoter and downstream of a potential ORF.

The bovine rotavirus g38 gene was cloned utilizing the METHOD FOR cDNACLONING BOVINE ROTAVIRUS g38 GENE. The bovine rotavirus g38 gene wasthen engineered to contain herpesvirus regulatory signals as shown inFIG. 20. This was accomplished by cloning the g38 gene BamHI fragmentcontained in pSY1053 between the BamHI and BglII sites in pSY1052. Theresulting plasmid, pSY1023, contained the PRV gX promoter in front ofthe g38 gene, and the HSV-1 TK polyadenylation signal behind the g38gene. The entire construct was flanked by XbaI sites to allow for theinsertion of the XbaI fragment into IBR by direct ligation.

S-IBR-004 was the starting virus for the generation of S-IBR-008.S-IBR-004 DNA and pSY1023 DNA were mixed together, cut with XbaI, andtransfected into rabbit skin cells according to the DIRECT LIGATIONPROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stockwas screened for recombinant virus by the ANTIBODY SCREEN FORRECOMBINANT HERPESVIRUS procedure using antibodies prepared against therotavirus g38 protein.

One of the viruses purified by this screen was S-IBR-008, which has thefollowing characteristics. It contains the rotavirus g38 gene plus theplasmid DNA inserted into the XbaI site in the long unique region of thevirus genome, but no longer contains the NEO gene of parent S-IBR-004 inthe unique short region. In fact, a small deletion was created in theunique short region at the location of the NEO gene, as evidenced by theabsence of an XbaI site at this location in S-IBR-008.

S-IBR-008 was shown to be expressing the rotavirus g38 gene by analysisof RNA transcription in infected cells, and by the ANTIBODY SCREEN FORRECOMBINANT HERPESVIRUS procedure using antibodies specific for the g38gene. S-IBR-008 was deposited on Jun. 18, 1986 pursuant to the BudapestTreaty on the International Deposit of Microorganisms for the Purposesof Patent Procedure with the Patent Culture Depository of the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852U.S.A. under ATCC Accession No. VR 2141. The structure of S-IBR-008 isshown in FIG. 20.

Example 16

S-IBR-018

S-IBR-018 is an IBR virus that has three foreign genes inserted: the E.coli beta-galactosidase gene and the neomycin resistance gene in theXbaI site in the unique long region, and the parainfluenza type 3 (PI-3)virus (ATCC No. VR-281) hemagglutinin gene (HN) in the HindIII site inthe unique long region adjacent to the Xba I site. The Xba I site islocated in the intergenic region upstream of the lactency-relatedtranscripts promoter and downstream of a potential ORF. The Hind IIIsite is located within a potential ORF upstream of the latency-relatedtranscripts.

For cloning the PI-3 HN gene, the SF-4 strain of PI-3 was grown inMADIN-DARBY bovine kidney (MDBK) cells in culture and RNA was extractedfrom infected cells. The RNA was used in a reverse transcriptionprotocol as outlined in the cDNA CLONING procedure using poly-dT asprimer for reverse transcriptase. From this procedure, a series ofclones was obtained that comprised parts of the genome of the PI-3virus. The location of the gene for the human PI-3 HN gene has beenpublished (25,26) and this information was used to locate the gene inapplicants' bovine PI-3 clones. The entire open reading frame of thebovine PI-3 HN gene was sequenced by applicants and is given in FIGS.21A-21B.

The HSV ICP4 promoter was used to express the PI-3 HN gene and the HSVTK poly-A signal was used to terminate transcription. The engineering ofthis construct was done as shown in FIGS. 22A-22C. The constructcontained (5′ to 3′) the HSV ICP4 promoter, the ICP4 TATA box, the ICP4cap site, a fusion within the ICP4 5′ untranslated region to the PI-3 HNgene at the HhaI site, the HN gene start codon, the HN structural gene,the HN stop codon, a fusion within the HN 3′ untranslated region to theHSV TK untranslated 3′ region, and the HSV TK poly-A signal sequence.

This plasmid also contained the beta-galactosidase (lacZ) gene under thecontrol of the PRV gX promoter with the gX poly-A termination signal, aswell as the neomycin resistance gene under the control of the gXpromoter with the TK poly-A termination signal. These latter two geneswere cloned in tandem at the XbaI site in BamHI-C fragment (FIGS.22A-22C). This BamHI-C fragment contained the homology regions for usein the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure.After the transfection step in the procedure, the resulting recombinantvirus from the transfection stock was selected for by the SELECTION OFG418 RESISTANT IBR VIRUS procedure, followed by the BLUOGAL™ SCREEN FORRECOMBINANT HERPESVIRUS procedure, and subsequently analyzed for theinsertion of the PI-3 HN gene by the SOUTHERN BLOTTING OF DNA procedure.The virus that resulted from this screening was designated S-IBR-018.

S-IBR-018 was deposited on Jul. 21, 1987 pursuant to the Budapest Treatyon the International Deposit of Microorganisms for the Purposes ofPatent Procedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. VR 2180. The structure of S-IBR-018 is shown inFIGS. 22A-22C.

Example 17

S-IBR-019

S-IBR-019 is an IBR virus that has three foreign genes inserted: the E.coli beta-galactosidase (lacZ) gene and the neomycin resistance gene inthe XbaI site in the unique long region, and the parainfluenza type 3(PI-3) virus fusion gene (F) in the HindIII site in the long uniqueregion adjacent to the XbaI site.

For cloning the PI-3 F gene, the SF-4 strain of PI-3 was grown in MDBKcells in culture and RNA was extracted from infected cells. The RNA wasused in a reverse transcription protocol as outlined in the cDNA CLONINGprocedure using poly-dT as primer for reverse transcriptase. From thisprocedure, a series of clones was obtained that comprised parts of thegenome of the PI-3 virus. The location of the gene for the Sendai virusF gene has been published (27) and this comparative sequence informationwas used to locate the homologous gene in applicants' bovine PI-3clones.

The HSV alpha-4 promoter was used to express the PI-3 F gene and the HSVTK poly-A signal was used to terminate transcription. The constructcontained (5′ to 3′) the HSV alpha-4 promoter, the alpha-4 TATA box, thealpha-4 cap site, a fusion in the alpha-4 5′ untranslated region to thePI-3 F gene, the F start codon, the F structural gene, the F stop codon,a fusion in the F 3′ untranslated region to the HSV TK 3′ untranslatedregion, and the TK poly-A signal sequence.

This plasmid also contained the beta-galactosidase (lacZ) gene under thecontrol of the PRV gX promoter with the gX poly-A termination signal, aswell as the neomycin resistance gene under the control of the gXpromoter with the TK poly-A termination signal. These latter two geneswere cloned in tandem at the XbaI site in BamHI-C fragment (FIGS.23A-23C). This BamHI-C fragment contained the homology regions for usein the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure.After the transfection step in the procedure, the resulting recombinantvirus from the transfection stock was selected for by the SELECTION OFG418 RESISTANT HERPESVIRUS procedure, followed by the BLUOGAL™ SCREENFOR RECOMBINANT HERPESVIRUS procedure, and subsequently analyzed for theinsertion of the PI-3 F gene by SOUTHERN BLOTTING OF DNA procedure. Thevirus that resulted from this screening was designated S-IBR-019.

The structure of S-IBR-019 is shown in FIGS. 23A-23C.

Example 18

S-IBR-032

S-IBR-032 is an IBR virus that has two foreign genes inserted: theEscherichia coli beta-galactosidase (lacZ) gene with the bovine viraldiarrhea virus (BVDV) g53 gene fused to the lacZ C-terminus and insertedin the long unique region at the XbaI restriction endonuclease site.

For cloning the BVDV g53 gene, the Singer strain of BVDV was grown inMADIN-DARBY bovine kidney (MDBK) cells in culture and the RNA wasextracted from infected cells. The RNA was used in a reversetranscriptase procedure as outlined in the cDNA CLONING procedure usingrandom primers for reverse transcriptase. From this procedure, a seriesof clones was obtained that comprised parts of the genome of BVDV. Thelocation of the gene for BVDV g53 has been published (66) and thiscomparative sequence information was used to locate the homologous genein the applicant's BVDV clones.

The PRV gX promoter was used to express lacZ with a region of BVDV g53fused to the C-terminus, and the PRV poly-A signal was used to terminatetranscription. A plasmid construct was engineered that contained (5′ to3′) the PRV gX promoter and then the coding region consisting of aminoacid codons 1-7 of the PRV gX gene, 10-1024 of the Escherichia coli lacZgene, and 684-758 of the BVDV major open reading frame, and the PRVpoly-A sequence. This lacZ fusion gene cassette was then excised fromthe plasmid vector at the flanking XbaI sites and cloned into the uniqueXbaI site in IBR-002 using the in vitro ligation method described inCONSTRUCTION OF DELETION VIRUSES. After the transfection step in DNATRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure, the resultingrecombinant virus was screened and isolated from the transfection stockusing the BLUOGAL™ SCREEN FOR RECOMBINANT HERPESVIRUS procedure, andsubsequently analyzed for the insertion of the BVDV g53 region bySOUTHERN BLOTTING OF DNA procedure. The virus that resulted from thisscreening was designated S-IBR-032.

Example 19

S-IBR-039

S-IBR-039 is an IBR virus that has three deletions in the short uniqueregion of the genome. The first deletion is approximately 2500 basepairs and begins in the HindIII K fragment approximately 1750 base pairsdownstream of the HindIII O/HindIII K junction and extends back throughthat junction. This deletion removes the US2 gene. The second deletionis approximately 1230 base pairs and begins in the HindIII K fragmentapproximately 3900 base pairs downstream of the HindIII O/Hind Kjunction and extends back toward that junction. This deletion removesamino acids 1 to 361 of the gG gene. The third deletion is approximately1410 base pairs and removes amino acids 77-547 of the gE gene.

S-IBR-039 was derived from S-IBR-037. This was accomplished utilizingthe homology vector 536-03.5 (see MATERIALS AND METHODS) and virusS-IBR-037 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS. The transfection stock was screened by theSCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES.The result of blue plaque purification was the recombinant virusdesignated S-IBR-039. This virus was characterized by restrictionmapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmedthe insertion of the β-galactosidase (lacZ) marker gene and the deletionof approximately 1230 base pairs of the gG gene. It was also confirmedthat an approximately 1410 base pair deletion has occurred in the regionof the gE gene (see above).

S-IBR-039 contains a deletion in the IBRV US2 gene which not onlyattenuates the virus but also has an unexpected effect of rendering thevirus fetal safe. Therefore, S-IBR-039 can be formulated into a vaccinewhich is superior from other IBRV vaccines in that in addition to beingsafe and effective in protecting cattle from infections with IBR virus,it is also safe for use in pregnant animals.

Such vaccine comprises an effective immunizing amount of S-IBR-039 and asuitable carrier. This vaccine may contain either inactivated or liveinfectious bovine rhinotracheitis virus S-IBR-039.

Suitable carriers for the infectious bovine rhinotracheitis virus arewell known in the art and include proteins, sugars, etc. One example ofsuch a suitable carrier is a physiologically balanced culture mediumcontaining one or more stabilizing agents such as stabilized, hydrolyzedproteins, lactose, etc.

In general, the vaccine of this invention contains an effectiveimmunizing amount of S-IBR-039 virus from about 10³ to 10⁸ PFU/dose.Preferably, the effective immunizing amount is from about 10⁴ to 10⁷PFU/dose for the live vaccine. Preferably, the live vaccine is createdby taking tissue culture fluids and adding stabilizing agents such asstabilized, hydrolyzed proteins. Preferably, the inactivated vaccineuses tissue culture fluids directly after inactivation of the virus.

The present invention also provides a method of immunizing an animal,particularly a bovine, against disease caused by infectious bovinerhinotracheitis virus which comprises administering to the animal aneffective immunizing dose of the vaccine comprising S-IBR-039. Thevaccine may be administered by any of the methods well known to thoseskilled in the art, for example, by intramuscular, subcutaneous,intraperitoneal or intravenous injection. Alternatively, the vaccine maybe administered intranasally or orally.

Another notable characteristic of S-IBR-039 is that it containsdeletions in the gG and gE genes so that no functional gG or gE isproduced upon viral replication. Said deletions in gG and gE, therefore,provides two negative serological markers for differentiating the virusfrom naturally-occurring IBR virus.

Accordingly, the present invention also provides a method fordistinguishing an animal vaccinated with the infectious bovinerhinotracheitis virus S-IBR-039 from an animal infected withnaturally-occurring infectious bovine rhinotracheitis virus. This methodcomprises analyzing a sample of a body fluid from the animal for thepresence of IBRV gG or gE and at least one other antigen which isnormally expressed in an animal infected by a naturally-occurringinfectious bovine rhinotracheitis virus and determining whether theantigen and gG or gE are present in the body fluid. The presence of theantigen and the absence of gG or gE in the body fluid is indicative ofan animal vaccinated with the vaccine and not infected with anaturally-occurring infectious bovine rhinotracheitis virus.

The presence of the antigen and of gG or gE in the body fluid may bedetermined by various methods, for example, by detecting in the bodyfluid antibodies specific for the antigen and for gG or gE.

S-IBR-039 has been deposited pursuant to the Budapest Treaty on theInternational Deposit of Microorganisms for the Purposes of PatentProcedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.

A study was conducted to determine the serological and clinical responseof young calves following intramuscular administration of S-IBR-037 andS-IBR-039. These results are presented in Table 7. Three calves wereinoculated intramuscularly with 10⁷ PFU of S-IBR-037 and S-IBR-039. Allcalves inoculated with S-IBR-037 and S-IBR-039 developed significantneutralizing antibody to IBR virus above the antibody levels of thecontact control. Attenuation of S-IBR-039 was demonstrated by theabsence of clinical signs in animals inoculated with the S-IBR-039virus.

TABLE 7 Serum Neutralizing Antibody Titers in Young Calves FollowingVaccination with S-IBR-037 and S-IBR-039 Serum Antibody Titer^(a) VirusDays Post Inoculation Construct Calf # 0 14 28 S-IBR-037 01 <2 9 5 06 <216 16 17 <2 5 7 Mean <2 10.0 9.3 S-IBR-039 11 <2 5 4 14 <2 12 9 07 <2 164 Mean <2 11.0 5.7 Control Mean <2 <2 <2 ^(a)Expressed as reciprocal ofdilution

Example 20

S-IBR-045

S-IBR-045, a recombinant IBR virus with deletions in the Tk, US2, gG andgE genes may be constructed in the following manner. S-IBR-045 would bederived from S-IBR-039 (see example 19) through the construction of twointermediate viruses. The first intermediate virus, S-IBR-043, would beconstructed utilizing the homology vector 591-46.12 (see MATERIALS ANDMETHODS) and virus S-IBR-039 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock would bescreened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATICMARKER GENES for a white plaque recombinant virus (uida substrate). Theresulting virus would have deletions of the Tk, US2, gG and gE genes andinsertion of lacZ gene in the gE gene deletion. Finally, S-IBR-045 wouldbe constructed, utilizing the homology vector 523-78.72 (see MATERIALSAND METHODS) and virus S-IBR-044 in the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stockwould be screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSINGENZYMATIC MARKER GENES for a white plaque recombinant virus (lacZsubstrate). This virus will be useful as a vaccine to protect cattlefrom infection with IBR. The combination of deletions will provide theappropriate attenuation which is required for a superior vaccine. Thisvirus will also provides two negative serological markers which may beused to distinguish vaccinated from infected animals. The availabilityof two negative markers permits one marker to be used as a confirmatorytest, greatly increasing the reliability of such a diagnosticdetermination.

Example 21

S-IBR-046

S-IBR-046, a recombinant IBR virus with deletions in the Tk, US2, gG andgE genes and the bovine viral diarrhea virus g53 gene inserted in placeof the gE gene, may be constructed in the following manner. S-IBR-046would be derived from S-IBR-044 (see example 20). It would beconstructed utilizing the homology vector 523-78.72, into which thebovine viral diarrhea virus g53 gene has been inserted, and virusS-IBR-044 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS. Note that the bovine diarrhea virus gene wouldbe cloned using techniques described in the methods section. The g53gene would be placed under the control of the HCMV immediate earlypromoter. The transfection stock would be screened by the SCREEN FORRECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES for a whiteplaque recombinant virus (lacZ substrate). This virus will be useful asa vaccine to protect cattle from infection with IBR virus and bovineviral diarrhea virus.

Example 22

S-IBR-047

S-IBR-047, a recombinant IBR virus with deletions in the Tk, US2, gG andgE genes and the parainfluenza type 3 genes for hemagglutinin and fusionprotein inserted in place of the gE gene may be constructed in thefollowing manner. S-IBR-047 would be derived from S-IBR-044 (see example20). It would be constructed utilizing the homology vector 523-78.72,into which the parainfluenza type 3 virus hemagglutinin and fusion geneshas been inserted, and virus S-IBR-044 in the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Note that theparainfluenza type 3 virus genes would be cloned using techniquesdescribed in the methods section. The transfection stock would bescreened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATICMARKER GENES for a white plaque recombinant virus (lacZ substrate). Thisvirus will be useful as a vaccine to protect cattle from infection withIBR virus and parainfluenza type 3 virus.

Example 23

S-IBR-049

S-IBR-049, a recombinant IBR virus with deletions in the Tk, US2, gG andgE genes and the bovine respiratory syncytial virus genes for theattachment, nucleocapsid and fusion proteins inserted in place of the gEgene may be constructed in the following manner. S-IBR-049 would bederived from S-IBR-044 (see example 20). It would be constructedutilizing the homology vector 523-78.72, into which the bovinerespiratory syncytial virus attachment nucleocapsid and fusion genes hadbeen inserted and virus S-IBR-044 in the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Note that the bovinerespiratory syncytial virus genes would be cloned using techniquesdescribed in the methods section. The attachment protein gene would beplaced under the control of the HCMV immediate early promoter and thefusion and nucleocapsid protein genes would be placed under the PRV gXpromoter. The transfection stock would be screened by the SCREEN FORRECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES for a whiteplaque recombinant virus (lacZ substrate). This virus will be useful asa vaccine to protect cattle from infection with IBR virus and bovinerespiratory syncytial.

Example 24

S-IBR-051

S-IBR-051, a recombinant IBR virus with deletions in the Tk, US2, gG andgE genes and the Pasteurella haemolytica genes for the leukotoxin andiron regulated outer membrane proteins inserted in place of the gE gene,may be constructed in the following manner. S-IBR-051 would be derivedfrom S-IBR-044 (see example 20). It would be constructed utilizing thehomology is vector 523-78.72, into which the Pasteurella haemolyticaleukotoxin and iron regulated outer membrane protein genes had beeninserted, and virus S-IBR-044 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUS. Note that the Pasteurellahaemolytica genes would be cloned using the techniques described in themethods section. The leukotoxin gene would be placed under the controlof the HCMV immediate early promoter and the iron regulated outermembrane protein genes would be placed under the PRV gX promoter. Thetransfection stock would be screened by the SCREEN FOR RECOMBINANTHERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES for a white plaquerecombinant virus (lacz substrate). This virus will be useful as avaccine to protect cattle from infection with IBR virus and Pasteurellahaemolytica.

Example 25

S-IBR-052

S-IBR-052 is an IBR virus that has three deletions in the unique shortregion of the genome. The first deletion is approximately 2500 basepairs and begins in the HindIII K fragment approximately 1750 base pairsdownstream of the HindIII O/HindIII K junction and extends back throughthat junction. This deletion removes the US2 gene. The second deletionis approximately 1230 base pairs and begins in the HindIII K fragmentapproximately 3900 base pairs downstream of the HindIII O/HindIII Kjunction and extends back toward that junction. This deletion removesamino acids 1 to 361 of the gG gene. The third deletion is approximately1410 base pairs and removes amino acids 77-547 of the gE gene.

S-IBR-052 was derived from S-IBR-039. This was accomplished utilizingthe homology vector 523-78.72 (see Materials and Methods) and virusS-IBR-039 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS. The transfection stock was screened by theBLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. The result of white plaquepurification was the recombinant virus designated S-IBR-052. This viruswas characterized by restriction mapping and by PCR analysis. Thisanalysis confirmed the deletion of the β-galactosidase (lacZ) markergene and the deletion of approximately 1410 base pairs of the gE gene.It was also confirmed that deletions present in the parent S-IBR-039virus were present in S-IBR-052.

S-IBR-052 contains a deletion in the IBRV US2 gene which not onlyattenuates the virus but also has an unexpected effect of rendering thevirus fetal safe. Therefore, S-IBR-052 can be formulated into a vaccinewhich is superior from other IBRV vaccines in that in addition to beingsafe and effective in protecting cattle from infections with IBR virus,it is also safe for use in pregnant animals.

Another notable characteristic of S-IBR-052 is that it containsdeletions in the gG and gE genes so that no functional gG or gE isproduced upon viral replication. Said deletions in gG and gE, therefore,provides two negative serological markers for differentiating the virusfrom wild-type virus.

S-IBR-052 on Feb. 4, 1994 was deposited pursuant to the Budapest Treatyon the International Deposit of Microorganisms for the Purposes ofPatent Procedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. VR 2443.

A study evaluating the safety of the S-IBR-052 vaccine demonstrates theabsence of a clinical response in young calves following intranasalvaccination with S-IBR-052 when compared to S-IBR-000 (Cooper strain).Four weaned, IBRV antibody negative calves per group were inoculatedintranasally with 2×10⁵ PFU of S-IBR-052 or 4×10⁵ PFU of S-IBR-000(Cooper strain). Calves were observed daily over a 4-week period forincreased body temperatures, respiratory disease, and ulcers of thenasal mucosa. As shown in Table 8, S-IBR-000 (Cooper) showed significantclinical signs whereas the S-IBR-052 showed no fibrile signs or nasalulcers and a reduced duration of respiratory signs. Since intranasal isthe natural route of infection for IBR virus, it is significant thatsignificant reduction in clinical signs are shown during S-IBR-052vaccination compared to S-IBR-000 (Cooper) vaccination.

TABLE 8 Clinical Response of Young Calves Following Intramuscular andIntranasal Vaccination with S-IBR-052 Febrile Repiratory Nasal SignsSigns Ulcers Virus Route of (Days (Numbers of (Number of ConstructInnoculati >104.5 F. ° days) days) S-IBR-000 Intranasal 4.7 20 4(Cooper) S-IBR-052 Intranasal .75 12 0

Example 26

Shipping Fever Vaccine

Shipping fever or bovine respiratory disease (BRD) complex is manifestedas the result of a combination of infectious diseases of cattle andadditional stress related factors (70). Respiratory virus infections,augmented by pathophysiological effects of stress, alter thesusceptibility of cattle to Pasteurella organisms by a number ofmechanisms. Control of the viral infections that initiate BRD as well ascontrol of the terminal bacterial pneumonia is essential to preventingthe disease syndrome (71).

The major infectious disease pathogens that contribute to BRD includebut are not limited to infectious bovine rhinotracheitis virus (IBRV),prarinfluenza type 3 virus (PI-3), bovine viral diarrhea virus (BVDV),bovine respiratory syncytial virus (BRSV), and Pasteurella haemolytica(71). Through out this application, applicants have disclosed examplesof recombinant IBR viruses that can be used as a vaccine to immunizeanimals against the various components of BRD.

The present invention also encompasses vaccines which are directed notonly to one particular component of BRD but to a combination of severalcomponents responsible for the disease, so that the array of pathogensresponsible for BRD can be controlled with a single immunization.

For example, a vaccine directed to several pathogens responsible for BRDcan be formulated as follows: first, the various IBRV vectored antigensfrom BRSV, PI-3, BVDV and P. haemolytica can be combined in a singlevaccine dose; secondly, the individual antigens from BRSV, PI-3, BVDVand P. haemolytica can be simultaneously cloned into the same IBR virusbackbone.

A preferred embodiment of the IBR virus. backbone for vectoring one ormore antigens from BRSV, PI-3, BVDV and P. haemolytica are S-IBR-039(see example 19) and S-IBR-052(see example 25), both of which containdeletions of the US2, gG, and gE genes.

S-IBR-039 is particularly appropriate as a backbone virus for purposesof vectoring said antigens. S-IBR-039 is a superior vaccine sincedeletions of the US2 gene provides the unexpected property of fetalsafety when used to vaccinate pregnant animals. In addition, deletionsof the gG and gE genes provide multiple negative markers useful indistinguishing vaccinated from infected animals.

Using S-IBR-039 virus as a backbone, the following viruses have beenconstructed which contains BRSV, PI-3, or BVDV antigens:

S-IBR-053

S-IBR-053 has been constructed by inserting the gene for BVDV g53 intothe gE deletion site of S-IBR-039. Expression of the BVDV g53 proteinfrom S-IBR-053 grown in cell culture has been confirmed byimmunofluorescence assay indicating that the correct immune reactiveepitope of BVDV g53 is present.

S-IBR-054

S-IBR-054 has been constructed by inserting the genes for PI-3 F and HNinto the gE deletion site of S-IBR-039. Expression of the PI-3 HNprotein from S-IBR-054 grown in cell culture has been confirmed byimmunofluorescence assay indicating that the correct immune reactiveepitope of PI-3 HN is present.

S-IBR-055

S-IBR-055 has been constructed by inserting the gene for BRSV F and Ninto the gE deletion site and inserting the gene for BRSV G into the gGdeletion site of the S-IBR-039 backbone.

S-IBR-059

S-IBR-059 has been constructed by inserting the gene for PI-3 F and HNinto the gG deletion site and inserting the gene for BVDV g53 into thegE deletion site of the S-IBR-039 backbone.

IBR viruses designated S-IBR-053, S-IBR-054, S-IBR-055 and S-IBR-059 arepresented as examples of recombinant IBR viruses containing one or moreantigens from PI-3, BVDV, BRSV, and Pasteurella haemolytica, which havebeen constructed using S-IBR-039 as a backbone virus. Applicants'present invention extends beyond these examples to cover any recombinantIBRV containing one or more antigens from PI-3, BVDV, BRSV andPasteurella haemolytica which is constructed using S-IBR-039 orS-IBR-052 as a backbone virus. The antigens to be inserted is selectedfrom the following group: PI-3 HN and F, BVDV g53, BRSV F, N and G, andPasteurella haemolytica leukotoxin. The following sites in S-IBR-039 orS-IBR-052 are used as insertion sites for these antigens: the gEdeletion site, gG deletion site, or US2 deletion site of S-IBR-039 orS-IBR-052; Hind III or Xba I sites within the unique long region of IBRVcontained on a 3900 base pair Apa I fragment (Homology vector 691-096.2)within the bamHI C fragment of IBRV. The Xba I site is located in theintergenic region upstream of the latency-related transcripts promoterand downstream of a potential ORF. The Hind III site is located within apotential ORF upstream of the latency-related transcripts; or 500 basepair EcoRV deletion within the repeat region of IBRV. Note that if acombination of antigens are inserted into one or more backbone viruses,this limits the number of IBR viruses required for BRD protection.

The following are several examples of how antigens from PI-3, BVDV, andBRSV are inserted into a backbone IBR virus, such as the S-IBR-039backbone and the S-IBR-052 backbone.

1. Genes encoding BVDV g53, PI-3 HN and F, and BRSV F, N, and G areinserted in combination into the S-IBR-039 backbone or the S-IBR-052backbone.

2. Genes encoding BVDV g53, BRSV F, N, and G are inserted in combinationinto the S-IBR-039 backbone or the S-IBR-052 backbone.

3. Genes encoding BRSV F, N, and G and PI-3 HN and F are inserted incombination into the S-IBR-039 backbone or the S-IBR-052 backbone.

Each of the above three IBR viruses are engineered further to includePasteurella haemolytica leukotoxin.

The viruses described through out this section (example 26) can be usedin combination as a vaccine against BRD. In addition, conventionallyderived vaccines (killed virus, inactivated bacterins and modified liveviruses) could be included with the recombinant multivalent vaccines aspart of the BRD vaccine formulation should such vaccine components proveto be more effective.

The present invention also provides a method for distinguishing ananimal vaccinated with the vaccine comprising the infectious bovinerhinotracheitis viruses described in this section (example 26). Thismethod comprises analyzing a sample of a body fluid from the animal forthe presence of gG or gE and at least one other antigen normallyexpressed in an animal infected by a naturally-occurring infectiousbovine rhinotracheitis virus. The presence of the antigen and theabsence of gG or gE in the body fluid is indicative of an animalvaccinated with the vaccine and not infected with a naturally-occurringIBR virus.

The presence of the antigen and of gG or gE in the body fluid may bedetermined by various methods, for example, by detecting in the bodyfluid antibodies specific for the antigen and for gG or gE.

Example 27

S-IBR-086

S-IBR-086 is an IBR virus that has three deletions in the short uniqueregion of the genome and two foreign genes inserted into the unique longregion of the genome. The deletions in the unique short are in the US2gene, the gG gene, and the gE gene. The extent of these deletions areidentical to those described for S-IBR-052 (Example 24). The genesfor-the bovine viral diarrhea virus (BVDV) glycoprotein 53 (g53) (amnoacids 1-394) under the control of the HCMV immediate early promoter andthe E. coli uida under the control of the PRV gX promoter were insertedinto a HindIII site within the BamHI C fragment of the IBRV genome (FIG.27).

S-IBR-086 was derived from S-IBR-052. This was accomplished utilizingthe homology vector 746-21.4 and virus S-IBR-052 in the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Homologyvector 746-21.4 was constructed by inserting the HCMV IE promoter BVDVg53 gene and PRV gX promoter uida gene into the unique HindIII site ofhomology vector 691-096.2 (see Materials and Methods). The BVDV g53 genewas isolated by CLONING OF BOVINE VIRAL DIARRHEA VIRUS g53 and g48GENES. The transfection stock was screened by the SCREEN FOR RECOMBINANTHERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The result of blue plaquepurification was the recombinant virus designated S-IBR-086. This viruswas characterized by restriction mapping and by PCR analysis. Thisanalysis confirmed the insertion of the β-glucuronidase (uidA) markergene and the insertion of the BVDV g53 gene. Expression of the BVDV g53was confirmed by immunoprecipitation using mouse monoclonal antibody toBVDV g53 protein. It was also confirmed that deletions present in theparent S-IBR-052 virus were present in S-IBR-086.

This virus is useful as a vaccine to protect cattle from infection withIBR virus and BVD virus. The deletion of the US2 gene attenuates thevirus, and the deletions of the glycoproteins G and E genes from thisvirus provides two negative serological markers for distinguishingvaccinated from infected animals.

S-IBR-069

S-IBR-069 is an IBR virus that has three deletions in the short uniqueregion of the genome and two foreign genes inserted into the unique longregion of the genome. The deletions in the unique short are in the US2gene, the gG gene, and the gE gene. The extent of these deletions areidentical to those described for S-IBR-052 (Example 24). The genes forthe bovine viral diarrhea virus (BVDV) glycoprotein 53 (g53) (aminoacids 1-394) under the control of the HCMV immediate early promoter,BVDV g48 gene (amino acid 1-226) under the control of the PRV gXpromoter, and the E. coli lacZ gene under the control of the HSV-1 TKpromoter were inserted into a HindIII site within the BamHI C fragmentof the IBRV genome (FIG. 27).

S-IBR-069 was derived from S-IBR-052. This was accomplished utilizingthe homology vector 756-15.5A and virus S-IBR-052 in the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Homologyvector 756-15.5A was constructed by inserting the HCMV IE promoter BVDVg53 gene, PRV gX promoter BVDV g48 gene and HSV-1 TK promoter lacZ geneinto the unique HindIII site of homology vector 691-096.2 (see Materialsand Methods). The BVDV g53 and g48 genes were isolated by CLONING OFBOVINE VIRAL DIARRHEA VIRUS g53 and g48 GENES. The transfection stockwas screened by the BLUOGAL™ SCREEN FOR RECOMBINANT HERPESVIRUS. Theresult of blue plaque purification was the recombinant virus designatedS-IBR-069. This virus was characterized by restriction mapping and byPCR analysis. This analysis confirmed the insertion of theβ-galactosidase (lacz) marker gene and the insertion of the BVDV g53gene and the BVDV g48 gene. Expression of the BVDV g53 was confirmed byimmunoprecipitation using mouse monoclonal antibody to BVDV g48 proteinIt was also confirmed that deletions present in the parent S-IBR-052virus were present in S-IBR-069.

This virus is useful as a vaccine to protect cattle from infection withIBR virus and BVD virus. The deletion of the US2 gene attenuates thevirus, an d the deletions of the glycoproteins G and E genes from thisvirus provides two negative serological markers for distinguishingvaccinated from infected animals.

S-IBR-074

S-IBR-074 is an IBR virus that has three deletions in the short uniqueregion of the genome and two foreign genes inserted into the unique longregion of the genome. The deletions in the unique short are in the US2gene, the gG gene, and the gE gene. The extent of these deletions areidentical to those described for S-IBR-052 (Example 24). The genes forthe bovine viral diarrhea virus (BVDV) glycoprotein 53 (g53) (aminoacids 1-394) under the control of the HCMV immediate early promoter,BVDV g48 gene (amino acids 1-226) under the control of the PRV gXpromoter, and the E. coli lacZ gene under the control of the HSV-1 TKpromoter were inserted into a HindIII site within the BamHI C fragmentof the IBRV genome (FIG. 27). The PRV gX signal sequence is fused inframe to the 5′ end of the BVDV g53 gene S-IBR-074 was derived fromS-IBR-052. This was accomplished utilizing the homology vector 756-35.38and virus S-IBR-052 in the HOMOLOGOUS RECOMBINATION PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS. Homology vector 756-35.38 wasconstructed by inserting the HCMV IE promoter PRV gX signal/BVDV g53gene, PRV gX promoter BVDV g48 gene and HSV-1 TK promoter lacZ gene intothe unique HindIII site of homology vector 691-096.2 (see Materials andMethods). The BVDV g53 and g48 genes were isolated by CLONING OF BOVINEVIRAL DIARRHEA VIRUS g53 and g48 GENES. The transfection stock wasscreened by the BLUOGAL™ SCREEN FOR RECOMBINANT HERPESVIRUS. The resultof blue plaque purification was the recombinant virus designatedS-IBR-074.

This virus is useful as a vaccine to protect cattle from infection withIBR virus and BVD virus. The deletion of the US2 gene attenuates thevirus, and the deletions of the glycoproteins G and E genes from thisvirus provides two negative serological markers for distinguishingvaccinated from infected animals.

Example 28

S-IBR-071

S-IBR-071 is an IBR virus that has three deletions in the short uniqueregion of the genome and two foreign genes inserted into the unique longregion of the genome. The deletions in the unique short are in the US2gene, the gG gene, and the gE gene. The extent of these deletions areidentical to those described for S-IBR-052 (Example 24). The genes forthe bovine respiratory syncytial virus (BRSV) fusion (F) gene (aminoacids 4-574) under the control of the HCMV immediate early promoter,BRSV attachment (G) gene (amnio acids 4-254) under the control of thePRV gX promoter, and the E. coli lacZ gene under the control of theHSV-1 TK promoter were inserted into a HindIII site within the BamHI Cfragment of the IBRV genome (FIG. 27).

S-IBR-071 was derived from S-IBR-052. This was accomplished utilizingthe homology vector 746-82.5B and virus S-IBR-052 in the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Homologyvector 746-82.SB was constructed by inserting the HCMV IE promoter BRSVF gene, PRV gX promoter BRSV G gene, and HSV-1 TK promoter lacZ geneinto the unique HindIII site of homology vector 691-096.2 (see Materialsand Methods). The BRSV F and G genes were isolated by CLONING OF BOVINERESPIRATORY SYNCYTIAL VIRUS FUSION PROTEIN AND NUCLEOCAPSID PROTEINGENES. The transfection stock was screened by the BLUOGAL™ SCREEN FORRECOMBINANT HERPESVIRUS. The result of blue plaque purification was therecombinant virus designated S-IBR-071.

This virus is useful as a vaccine to protect cattle from infection withIBR virus and BRSV. The deletion of the US2 gene attenuates the virus,and the deletions of the glycoproteins G and E genes from this virusprovides two negative serological markers for distinguishing vaccinatedfrom infected animals.

In an alternative embodiment of S-IBR-071 and S-IBR-072, a recombinantIBR virus is constructed that has three deletions (the US2 gene, the gGgene, and the gE gene) in the unique short and insertions of the BRSV Fand G genes and lacZ gene in the HindIII site in the unique long and theBRSV N gene and uidA gene in the EcoRV site in the repeat region. Therecombinant IBR viruses derived utilizing the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS from S-IBR-071 and ahomology vector containing the BRSV N gene and uida gene withappropriate promoters inserted at the EcoRV site of homology vector769-73.1 (see Materials and Methods). The transfection stock is screenedby the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKERGENES. The result of blue plaque purification is the recombinant IBRvirus as described.

S-IBR-072

S-IBR-072 is an IBR virus that has three deletions in the short uniqueregion of the genome and two foreign genes inserted into the unique longregion of the genome. The deletions in the unique short are in the US2gene, the gG gene, and the gE gene. The extent of these deletions areidentical to those described for S-IBR-052 (Example 24). The genes forthe bovine respiratory syncytial virus (BRSV) nucleocapsid (N) gene(amino acids 1-399) under the control of the PRV gX promoter, and the E.coli lacZ gene under the control of the HSV-1 TK promoter were insertedinto a EcoRV site within the BamHI C fragment in the repeat region ofthe IBRV genome (FIG. 27).

S-IBR-072 is derived from S-IBR-052. A homology vector is constructed byinserting PRV gX promoter BRSV N gene and HSV-1 TK promoter lacZ geneinto the unique EcoRV site of homology vector 769-73.1 (see Materialsand Methods). The BRSV N gene is isolated by CLONING OF BOVINERESPIRATORY SYNCYTIAL VIRUS FUSION PROTEIN AND NUCLEOCAPSID PROTEINGENES. S-IBR-072 is accomplished utilizing the homology vector and virusS-IBR-052 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS. The transfection stock is screened by theBLUOGAL™ SCREEN FOR RECOMBINANT HERPESVIRUS. The result of blue plaquepurification is the recombinant virus designated S-IBR-072.

This virus is useful as a vaccine to protect cattle from infection withIBR virus and BRSV. The deletion of the US2 gene attenuates the virus,and the deletions of the glycoproteins G and E genes from this virusprovides two negative serological markers for distinguishing vaccinatedfrom infected animals.

S-IBR-073

S-IBR-073 is an IBR virus that has three deletions in the short uniqueregion of the genome and two foreign genes inserted into the unique longregion of the genome. The deletions in the unique short are in the US2gene, the gG gene, and the gE gene. The extent of these deletions areidentical to those described for S-IBR-052 (Example 24). The genes forthe parainfluenza virus type 3 (PI-3) fusion (F) gene (amino acids4-540) under the control of the HCMV immediate early promoter, PI-3haemagglutinin/neuraminidase (HN) gene (amino acids 1-573) under thecontrol of the PRV gX promoter, and the E. coli lacZ gene under thecontrol of the HSV-1 TK promoter were inserted into the gG deletion sitewithin the unique short of the IBRV genome.

S-IBR-073 was derived from S-IBR-052. This was accomplished utilizingthe homology vector 756-11.17 and virus S-IBR-052 in the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The PI-3F and HN genes were isolated by CLONING OF PARAINFLUENZA-3 FUSION ANDHEMAGGLUTININ GENES. The transfection stock was screened by the BLUOGAL™SCREEN FOR RECOMBINANT HERPESVIRUS. The result of blue plaquepurification was the recombinant virus designated S-IBR-073.

This virus is useful as a vaccine to protect cattle from infection withIBR virus and PI-3 virus. The deletion of the US2 gene attenuates thevirus, and the deletions of the glycoproteins G and E genes from thisvirus provides two negative serological markers for distinguishingvaccinated from infected animals.

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78 1 39 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide primer derived from sequence of bovinerotavirus 1 gggaattctg caggtcacat catacaattc taatctaag 39 2 43 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide primer derived from sequence of bovine rotavirus 2gggaattctg caggctttaa aagagagaat ttccgtttgg cta 43 3 42 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotideprimer derived from sequence of bovine rotavirus 3 acgtcggatc ccttaccaaaccacgtctta ctcttgtttt cc 42 4 40 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide primer derived fromsequence of bovine rotavirus 4 acataggatc ccatgggaga aaacataacacagtggaacc 40 5 42 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide primer derived from sequence ofbovine rotavirus 5 cttggatcct catccatact gagtccctga ggccttctgt tc 42 631 DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide primer derived from sequence of bovine rotavirus 6catagatctt gtggtgctgt ccgacttcgc a 31 7 40 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide primerderived from sequence of bovine rotavirus 7 cgtcggatcc ctcacagttccacatcattg tctttgggat 40 8 41 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide primer derived fromsequence of bovine rotavirus 8 cttaggatcc catggctctt agcaaggtcaaactaaatga c 41 9 41 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide primer derived from sequence ofbovine rotavirus 9 cgttggatcc ctagatctgt gtagttgatt gatttgtgtg a 41 1041 DNA Artificial Sequence Description of Artificial SequenceSyntheticoligonucleotide primer derived from sequence of bovine rotavirus 10ctctggatcc tcatacccat catcttaaat tcaagacatt a 41 11 37 DNA ArtificialSequence Description of Artificial SequenceSynthetic oligonucleotideprimer derived from sequence of bovine rotavirus 11 tgcaggatcctcatttacta aaggaaagat tgttgat 37 12 35 DNA Artificial SequenceDescription of Artificial SequenceSynthetic oligonucleotide primerderived from sequence of bovine rotavirus 12 ctctggatcc tacagccatgaggatgatca tcagc 35 13 34 DNA Artificial Sequence Description ofArtificial SequenceSynthetic oligonucleotide primer derived fromsequence of bovine rotavirus 13 ttatggatcc tgctgctgtg ttgaacaact ttgt 3414 38 DNA Artificial Sequence Description of ArtificialSequenceSynthetic oligonucleotide primer derived from sequence of bovinerotavirus 14 ccgcggatcc catgaccatc acaaccataa tcatagcc 38 15 43 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide primer derived from sequence of bovine rotavirus 15cgtcggatcc cttagctgca gttttttgga acttctgttt tga 43 16 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotideprimer derived from sequence of bovine rotavirus 16 cataggatcccatggaatat tggaaacaca caaacagcac 40 17 31 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide primerderived from sequence of bovine rotavirus 17 tatagatctt agacttacaaccctaaaaaa c 31 18 36 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide primer derived from sequence ofbovine rotavirus 18 cgtggatcca actctataat gtgtgaaaca atatag 36 19 1079DNA Infections Bovine Rhinotracheitis Virus 19 ttaagcgttg ccgtggcggtcgccatggtg actatagtca cgtgtggccg gataggcgcg 60 gcgccttcca ggcaagcccagacgtgcgcc gcgcgggtgt ggcgtttcct tgccgagcag 120 agccgggcgc tgacggcaagccggctgggg acgacggtcg ttgtcttcga tcacgcccta 180 gtaaaaacgg cgaagggctgcacgtcgacg tcaacgtcaa gccagcggcg cgggtggctt 240 ttgtcgacac agcgcccttggcccgggcgc cggcttagcc cgccaccgcc aaccggcgag 300 tgggtcagct ggtcgacggctacaaacttg ctgaaactcg gccgcgcgag ggctcggccc 360 ttccacatgt gggtttttggcgccgccgat ttgtacgcgc ctatttttgc gcacattgcc 420 gccacgacgc gcttggtttacgcgcagctg gactgtacgt ttgcgggagc ggcgtggcgg 480 ctcccgcggc gcggcccggccatcgctagc ccgtggccgc cctacgatac cccgacactc 540 cctgagctgg tggccggtggtgtccttttc cggctggtct acgaagtcgt agaccgcggg 600 cggcgccccg ccccgccaaacgcgagcccc cgtgccccag gggctcgccc ccgcgcgcgc 660 catgtgctat cctttaaaggccgcacccag cgccggcgtt tggtcatttg ctttgtgacc 720 gcgccgaggg accatgttccgccagggcac ccccaaccgc gtggtgatca gcacagtgcc 780 gttgagcaga gaggcgaccgcgaccgcgac cgccggcacc ggtcccggat gcgagggggg 840 gcttggtggc tggcgactctttacagtgcc gccacgagca agaagacggc ctgtatgcta 900 tcgtcccgcc ggactattttccggtggtgc cctcgtccaa gcccctgctg gtgaaagttc 960 ccgctcccgg cgcgagtcccgaccgaactg ggggcgcagt tcactttgaa tgtgttcccg 1020 cgccgcgccg accgctgcagttctttcgtc agctttacga cggttcattc gttaagctt 1079 20 17 PRT ′AxialSeamount′ polynoid polychaete 20 Met Trp Val Phe Gly Ala Ala Asp Leu TyrAla Pro Ile Phe Ala His 1 5 10 15 Ile 21 17 PRT Herpes Simplex Virus 121 Leu Trp Val Val Gly Ala Ala Asp Leu Cys Val Pro Phe Leu Glu Tyr 1 510 15 Ala 22 17 PRT Parvovirus 22 Leu Trp Ile Leu Gly Ala Ala Asp LeuCys Asp Gln Val Leu Leu Ala 1 5 10 15 Ala 23 17 PRT Herpes Simplex Virus2 23 Leu Trp Val Val Gly Ala Ala Asp Leu Cys Val Pro Phe Phe Glu Tyr 1 510 15 Ala 24 19 PRT Marek′s Disease Virus 24 His Ser Leu Trp Ile Val GlyAla Ala Asp Ile Cys Arg Ile Ala Leu 1 5 10 15 Glu Cys Ile 25 60 DNAArtificial Sequence Description of Artificial Sequence IBRV CooperStrain 25 tgagcgcgcg ccgctgcatg ctggtgcgaa ctcacgccga gcgcgcgtgcgagcaagctt 60 26 60 DNA Artificial Sequence Description of ArtificialSequence IBRV Nasalgen Strain 26 ctagtaaaaa cggcgaaggg ctggtgcgaactcacgccga gcgcgcgtgc gagcaagctt 60 27 60 DNA Artificial SequenceDescription of Artificial Sequence IBRV Cooper Strain 27 ctagtaaaaacggcgaaggg ctgcacgtcg acgtcaacgt caagccagcg gcgcgggtgg 60 28 48 DNAArtificial Sequence Description of Artificial Sequence Synthetic LinkerSequences 28 gatttaggtg acactataga atacacggaa ttcgagctcg ccccatgg 48 2999 DNA Artificial Sequence Description of Artificial Sequence SyntheticLinker Sequences 29 ttaagtggga tcccggcgcg caggcgcgca cgtcggtcgcggtcgcgcgc catgggggat 60 cctctagagc ttgggctgca ggtcctgatt gatacactg 9930 99 DNA Artificial Sequence Description of Artificial SequenceSynthetic Linker Sequences 30 gccccgatcg tccacacgga gcgcggctgccgacacggat ctgatcaaga gacaggatga 60 ggatcgtttc gcatgattga acaagatggattgcacgca 99 31 100 DNA Artificial Sequence Description of ArtificialSequence Synthetic Linker Sequences 31 ggaccttgca caagatagcg tggtccggccaggacgacga ggcttgcagg atcctctaga 60 gtcgggagat gggggaggct aactgaaacacggaaggaga 100 32 124 DNA Artificial Sequence Description of ArtificialSequence Synthetic Linker Sequences 32 gtgttgctgc gttcccgacc tgcagcccaagctctagagt cgacctgcag cccaagctct 60 agagtcgacc tgcagcccaa gctcagatctgctcatgctc gcggccgcca tgcccccgga 120 agcg 124 33 60 DNA ArtificialSequence Description of Artificial Sequence Synthetic Linker Sequences33 aggcagatct gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc 6034 1386 DNA ′Axial Seamount′ polynoid polychaete 34 gcgatcatgcctgccgcccg gaccggcacc ttggccgccg tcgccctaat cctgctctgc 60 ggggccgccgttttgcggcc ccgcgcccga cgacctctgt ttcgccgacg tgcgccgcac 120 tggcatggcgccctcccgcc cgctggggcc cgtcctgaac ctagcggcct cggatttgac 180 ctcgcgggtttcggtgcgcg cggtggagct tcgcgcgctg cgccctggcc ctcttggaca 240 tggcggagacggtggtgccc ggcggaccgc gagccscacg tcgtcgacgt cggctgggct 300 taccaagacggggactgcat ggtgcctctg gcatatcgcc agtactttaa ctgcacgggg 360 ggcgcgctgcccggccaaaa cgtctgcgcc gggctctctg agacccgcat ccgcggtggc 420 tttggaacctccgactacgc gctctacggg acgtcgctag tactgcgccc cggcctgtac 480 gaccgcgggacctacatcta cttccttgga tacggcccag acgacatcta cgtgggcagc 540 gtcacgctcatggtgggcgc cgacatccac aaatacccct gcgggctgga ccgagggctc 600 ggtgtggccctgcaccacaa gagcggaccg gcccgacctc tgacagagga cgacgccacc 660 ggcgactgggcctgcggctg cttccccgcc cttgttgagg ttgacgcggt gtggggcaac 720 gtaagcgccgcagagctggg cctggccgac ccgatcgact acgccgacga agggggtgag 780 gtcgaagtgctcgaggacga agccgggagc gccagcggaa acctgccgca ggacgacccc 840 gaccccgacctcgcagattg ccggaccgtc gggctcttta gcgaaagcga catgttccgg 900 accgccagcgggcccgaatc gctgctgatc ggcgccgttg ccaaggacgt cctgacggtg 960 cccctcaatctgccgcccgg ccgctcttac gaggccctgc gaaacgcatc gctggagtgc 1020 aactcccgcccgcgcgagac cggcgacgca gcggtggtgg tgatgtctct ccaggagccc 1080 gctcgcctcgagcgccgccc cgatgcccgc gccaccgatc cggagtttgg gctctttggc 1140 ctgcccgatgaccccgccgt gcgcgcggca ttctcatcgg cctcgcgatc gctctgctgg 1200 tgctgctgtttcgctggtga tcgtgctcgt ctgcgcctgc cggctcgccc gcccagccaa 1260 ggctgcgcgacgccccgcgc cgccacgttc gccaagagca accccgcgta cgagccgatg 1320 ctcagcgtctgatcgccggc accccacgcc gccccgaccc cgctgtcccg cgtttacaat 1380 aaacag 138635 34 PRT Parvovirus 35 Val Gly Trp Ala Tyr Gln Asp Gly Asp Cys Met ValPro Leu Ala Tyr 1 5 10 15 Arg Gln Tyr Phe Asn Cys Thr Gly Gly Ala LeuPro Gly Asn Val Leu 20 25 30 Cys Ala 36 34 PRT Parvovirus 36 Val Ala TrpPhe Phe Asp Gly Gly His Cys Lys Val Pro Leu Val His 1 5 10 15 Arg GluTyr Tyr Gly Cys Pro Gly Asp Ala Met Pro Ser Val Glu Thr 20 25 30 Cys Thr37 34 PRT Herpes Simplex Virus - 2 37 Val Thr Tyr Tyr Arg Leu Thr ArgAla Cys Arg Gln Pro Ile Leu Leu 1 5 10 15 Arg Gln Tyr Gly Gly Cys ArgGly Gly Glu Pro Pro Ser Pro Lys Thr 20 25 30 Cys Gly 38 102 DNAArtificial Sequence Description of Artificial Sequence Synthetic LinkerSequences 38 cacatacgat ttaggtgaca ctatagaata caagcttggg ctgcaggtcgactctagagt 60 cgacctgcag tgaataataa aatgtgtgtt tgtccgaaat ac 102 39 102DNA Artificial Sequence Description of Artificial Sequence SyntheticLinker Sequences 39 gcgtttgaga tttctgtccc gactaaattc atgtcgcgcgatagtggtgt ttatcgccga 60 tagagatggc gatattggaa aaatcgatat ttgaaaatat gg102 40 102 DNA Artificial Sequence Description of Artificial SequenceSynthetic Linker Sequences 40 catattgaaa atgtcgccga tgtgagtttctgtgtaactg atcgcgtgtt tggaggcaac 60 cggggcctgc tcccgacggc cagcgacgacgtggtgctca ag 102 41 102 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Linker Sequences 41 atgtctctcc aggagcccgctcgcctcgag ggcctgccct cgcagctgcc cgtcttcgag 60 gacacgcagc gctacgacgcctcccccgcg tccgtgagct gg 102 42 102 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Linker Sequences 42 cccgtgagcagcatgatcgt cgtcatcgcc ggcatcggga tcctggccat cgtgctggtc 60 atccatatggcgatcatcag ggcccgggcc cggaacgacg gc 102 43 75 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Linker Sequences 43gggccagtac cggcgcctgg tgtccgtcga ctctagagtc gacctgcagc ccaagctttg 60gcgtaatcat ggtca 75 44 57 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Linker Sequences 44 acatacgatt taggtgacactatagaatac aagcttaacg aatgaaccgt cgtaaag 57 45 96 DNA ArtificialSequence Description of Artificial Sequence Synthetic Linker Sequences45 gtcgaagtgc tcgaaattcg agctcgcccg gggatcctct agagtcgacc tgcaggtgga 60ctctagagga tctcgacgga caccaggcgc cggtac 96 46 57 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Linker Sequences 46gggcggggcc gggtcagccg gatctagagt cccaggaccc aacgctgccc gagtttg 57 47 57DNA Artificial Sequence Description of Artificial Sequence SyntheticLinker Sequences 47 tcccagtcac gacgttgtaa aacgacggga tccatggtcccggtgtcttc tatggag 57 48 57 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Linker Sequences 48 attcactgca ggtcgactctagaggatccc cgggcgagct cgaatttcga gcgccgc 57 49 57 DNA ArtificialSequence Description of Artificial Sequence Synthetic Linker Sequences49 gcgcgcgcgt acaacgccac ggtcataggg cgagctcgaa ttcgtaatca tggtcat 57 5057 DNA Artificial Sequence Description of Artificial Sequence SyntheticLinker Sequences 50 atacacatac gatttaggtg acactataga atacaagctcgcgtgtttgg aggcaac 57 51 102 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Linker Sequences 51 tcggggtagc cccaattcgagctcgcccgg ggatcctcta gagtcgacct gcaggtcgac 60 tctagaggat ctcgacggacaccaggcgcc ggtactggcc ct 102 52 57 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Linker Sequences 52 gggcggggccgggtcagccg gatctagagt cccaggaccc aacgctgccc gagtttg 57 53 57 DNAArtificial Sequence Description of Artificial Sequence Synthetic LinkerSequences 53 tcccagtcac gacgttgtaa aacgacggga tccatggtcc cggtgtcttctatggag 57 54 57 DNA Artificial Sequence Description of ArtificialSequence Synthetic Linker Sequences 54 attcactgca ggtcgactct agaggatccccgggcgagct cgaatttcga gcgccgc 57 55 57 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Linker Sequences 55gcgcgcgcgt acaacgccac ggtcataggg cgagctcgaa ttcgtaatca tggtcat 57 56 57DNA Artificial Sequence Description of Artificial Sequence SyntheticLinker Sequences 56 atacacatac gatttaggtg acactataga atacaagctcgcgtgtttgg aggcaac 57 57 84 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Linker Sequences 57 cggggtagcc ccaattcgagctcgcccggg gatcctctag aggatccccg ggcgagctcg 60 aatttcgagc gccgccccgatgcc 84 58 57 DNA Artificial Sequence Description of Artificial SequenceSynthetic Linker Sequences 58 gcgcgcgcgt acaacgccac ggtcatagggcgagctcgaa ttcgtaatca tggtcat 57 59 2040 DNA IBRV 59 gcgggcaaggcggaggaaga ccgggggcag gagctgcgtg gagggcggag ccgttgagcg 60 gcccgaccgccgccgggttg ttaaatgggt ctcgcgcggc tcgtggttcc acaccgcgcc 120 ggagaaccagcgcgcagctt cgctgcgtgt gtcccgcgag ctgcgttccg gggaacggcg 180 cgcgcgagagggttcgaaaa gggcatttgg caatgcaacc caccgcgccg ccccggcssg 240 gttgcgccgctgctgctgcc gcagttattg cttttcgggc tgatggccga ggccaagccc 300 gcgaccgaaaccccgggctc ggcttcggtc gacacggtct tcacggcgcg cgctggcgcg 360 cccgtctttctcccagggcc cgcggcgcgc ccggacgtgc gcgccgttcg cggctggagc 420 gtcctcgcggccgcctgctc gccgcccgtg ccggagcccg tctgcctcga cgaccgcgag 480 tgcttcaccgacgtggccct ggacgcggcc tgcctgcgaa ccgcccgcgt ggccccgctg 540 gccatcgcggagctcgccga gcggcccgac tcaacgggcg acaaagagtt tgttctcgcc 600 gacccgcacgtctcggcgca gctgggtcgc aacgcgaccg gggtgctgat cgcggccgca 660 gccgaggaggacggcggcgt gtacttcctg tacgaccggc tcatcggcga cgccggcgac 720 gaggagacgcagttggcgct gacgctgcag gtcgcgacgg ccggcgcgca gggcgccgcg 780 cgggacgaggagagggaacc agcgaccggg cccacccccg gcccgccgcc ccaccgcacg 840 acgacacgcgcgcccccgcg gcggcacggc gcgcgcttcc gcgtgctgcc gtaccactcc 900 cacgtatacaccccgggcga ttcctttctg ctatcggtgc gtctgcagtc tgagtttttc 960 gacgaggctcccttctcggc cagcatcgac tggtacttcc tgcggacggc cggcgactgc 1020 gcgctcatccgcatatacga gacgtgcatc ttccaccccg aggcaccggc ctgcctgcac 1080 cccgccgacgcgcagtgcag cttcgcgtcg ccgtaccgct ccgagaccgt gtacagccgg 1140 ctgtacgagcagtgccgccc ggaccctgcc ggtcgctggc cgcacgagtg cgagggcgcc 1200 gcgtacgcggcgcccgttgc gcacctgcgt cccgccaata acagcgtaga cctggtcttt 1260 gacgacgcgccggctgcggc ctccgggctt tacgtctttg tgctgcagta caacggccac 1320 gtggaagcttgggactactg cctagtcgtt acttcggacc gtttggtgcg cgcggtcacc 1380 gaccacacgcgccccgaggc cgcagccgcc gacgctcccg agccaggccc accgctcacc 1440 agcgagccggcgggggsgcc caccgggccc gcgccctggc ttgtggtgct ggtgggcgcg 1500 cttggactcgcgggactggt gggcatcgca gccctcgccg ttcgggtgtg cgcgcgccgc 1560 gcaagccagaagcgcaccta cgacatcctc aaccccttcg ggcccgtata caccagcttg 1620 ccgaccaacgagccgctcga cgtggtggtg ccagttagcg acgacgaatt ttccctcgac 1680 gaagactcttttgcggatga cgacagcgac gatgacgggc ccgctagcaa cccccctgcg 1740 gatgcctacgacctcgccgg cgccccagag ccaactagcg ggtttgcgcg agcccccgcc 1800 aacggcacgcgctcgagtcg ctctgggttc aaagtttggt ttagggaccc gcttgaagac 1860 gatgccgcgccagcgcggac cccggccgca ccagattaca ccgtggtagc agcgcgactc 1920 aagtccatcctccgctaggc gccccccccc gcgcgctgtg ccgtctgacg gaaagcaccc 1980 gcgtgtagggctgcatataa atggagcgct cacacaaagc ctcgtgcggc tgcttcgaag 2040 60 720 DNAIBRV 60 gtggaagctt gggactactg cctagtcgtt acttcggacc gtttggtgcgcgcggtcacc 60 gaccacacgc gccccgaggc cgcagccgcc gacgctcccg agccaggcccaccgctcacc 120 agcgagccgg cgggggsgcc caccgggccc gcgccctggc ttgtggtgctggtgggcgcg 180 cttggactcg cgggactggt gggcatcgca gccctcgccg ttcgggtgtgcgcgcgccgc 240 gcaagccaga agcgcaccta cgacatcctc aaccccttcg ggcccgtatacaccagcttg 300 ccgaccaacg agccgctcga cgtggtggtg ccacttagcg acgacgaattttccctcgac 360 gaagactctt ttgcggatga cgacagcgac gatgacgggc ccgctagcaacccccctgcg 420 gatgcctacg acctcgccgg cgccccagag ccaactagcg ggtttgcgcgagcccccgcc 480 aacggcacgc gctcgagtcg ctctgggttc aaagtttggt ttagggacccgcttgaagac 540 gatgccgcgc cagcgcggac cccggccgca ccagattaca ccgtggtagcagcgcgactc 600 aagtccatcc tccgctaggc gccccccccc gcgcgctgtg ccgtctgacggaaagcaccc 660 gcgtgtaggg ctgcatataa atggagcgct cacacaaagc ctcgtgcggctgcttcgaag 720 61 50 PRT HSV-1 61 Trp Leu Arg Phe Asp Val Pro Thr SerCys Ala Glu Met Arg Ile Tyr 1 5 10 15 Glu Ser Cys Leu Tyr His Pro GlnLeu Pro Glu Cys Leu Ser Pro Ala 20 25 30 Asp Ala Pro Cys Ala Ala Ser ThrTrp Thr Ser Arg Leu Ala Val Arg 35 40 45 Ser Tyr 50 62 53 PRT PRV 62 TrpTyr Tyr Ala Arg Ala Pro Pro Arg Cys Leu Leu Tyr Tyr Val Tyr 1 5 10 15Glu Pro Cys Ile Tyr His Pro Arg Ala Pro Glu Cys Leu Arg Pro Val 20 25 30Asp Pro Ala Cys Ser Phe Thr Ser Pro Ala Ala Arg Ala Ala Leu Val 35 40 45Ala Arg Arg Ala Tyr 50 63 52 PRT VZV 63 Trp Leu Tyr Val Pro Ile Asp ProThr Cys Gln Pro Met Arg Leu Tyr 1 5 10 15 Ser Thr Cys Leu Tyr His ProAsn Ala Pro Gln Cys Leu Ser His Met 20 25 30 Asn Ser Gly Cys Thr Phe ThrSer Pro His Leu Ala Gln Arg Val Ala 35 40 45 Ser Thr Val Tyr 50 64 52PRT IBRV 64 Trp Tyr Phe Leu Arg Thr Ala Gly Asp Cys Ala Leu Ile Arg IleTyr 1 5 10 15 Glu Thr Cys Ile Phe His Pro Glu Ala Pro Ala Cys Leu HisPro Ala 20 25 30 Asp Ala Gln Cys Ser Phe Ala Ser Pro Tyr Arg Ser Glu ThrVal Tyr 35 40 45 Ser Arg Leu Tyr 50 65 85 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Linker Sequences 65ttgggctgca ggtcgactct agaggatccc ctatggtaca agatcgagag cgggtgccgc 60ccggccgctg tactacatgg agtac 85 66 84 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Linker Sequence 66 tccgggctttacgtctttgt gctgcagtac aacggccacg tggaagcttg ggactacagc 60 ctagtcgttacttcggaccg tttg 84 67 84 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Linker Sequences 67 ccttcaccgc cgccggaaggctccatcgtg tccatcccca tcctcgagct cgaattgggg 60 atcctctaga gtcgacctgcagcc 84 68 60 DNA Artificial Sequence Description of Artificial SequenceSynthetic Linker Sequences 68 ctatagaata cacggaattc gagctcgcccgggtgagcgg cctaggccct cccccgaccg 60 69 90 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Linker Sequences 69atggccgagg ccaagcccgc gaccgaaacc ccggggatcc tctagagtcg acgtctgggg 60cgcgggggtg gtgctcttcg agacgctgcc 90 70 90 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Linker Sequence 70acctttgcgc atctccacag ctcaacaatg aagtgggcaa cgtggatcga tcccgtcgtt 60ttacaacgtc gtgactggga aaaccctggc 90 71 201 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Linker Sequences 71tggagcccgt cagtatcggc ggaaatccag ctgagcgccg gtcgctacca ttaccagttg 60gtctggtgtc aaaaagatct agaataagct agaggatcga tcccctatgg cgatcatcag 120ggcccgatcc cctatggcga tcatcagggc ccgggcccgg aacgacggct accgccacgt 180ggcctccgcc tgacccggcc c 201 72 90 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Linker Sequences 72 ggcgcctggt gtccgtcgactctagagtcg acctgcagcc caagctctag caacccccct 60 gcggatgcct acgacctcgccggcgcccca 90 73 1880 DNA IBRV 73 aggaacaaag ttgttcaaca cagcagcagcgaacagaccc aaaggcagcg cagaggcgac 60 accgaaccca aaatggaata ttggaaacacacaaacagca caaaaaacac caacaatgaa 120 accgaaacaa ccagaggcaa acacagtagcaaggttacaa atatcataat gtacaccttc 180 tggacaataa catcaacaat attattagtcatttttataa tgatattgac aaacttaatt 240 caagagaaca atcataataa attaatgttgcaggaaataa gaaaagaatt cgcggcaata 300 gacaccaaga ttcagaggac ctcggatgacattggaacct caatacagtc aggaataaat 360 acaagacttc tcacaattca gagtcatgttcaaaactata tcccactatc actaacacaa 420 caaatgtcag atctcagaaa atttatcaatgatctaacaa ataaaagaga acatcaagaa 480 gtgccaatac agagaatgac tcatgatagaggtatagaac ccctaaatcc agacaagttc 540 tggaggtgta catctggtaa cccatctctaacaagtagtc ctaagataag gttaatacca 600 gggccaggtt tattagcaac atctactacagtaaatggct gtattagaat cccatcgtta 660 gcaatcaatc atttaatcta cgcttacacctctaatctta tcacccaggg ctgtcaaaat 720 atagggaaat cttaccaagt actacaaatagggataatta ctataaattc ggacctagta 780 cctgatttaa atcccagagt cacacatacatttaatattg atgataatag gaaatcttgc 840 tctctggcac tattgaatac agatgtttatcagttatgct caacaccaaa agttgatgag 900 agatccgatt atgcatcaac aggtattgaggatattgtac ttgacattgt cactaataat 960 ggattaatta taacaacaag gtttacaaataataatataa cttttgataa accgtatgca 1020 gcattgtatc catcagtagg accaggaatctattataagg gtaaagttat ctttctcgga 1080 tatggaggtc tagagcatga agaaaacggagacgtaatat gtaatacaac tggttgtcct 1140 ggcaaaacac agagagactg taatcaggcttcttatagcc catggttctc aaataggaga 1200 atggtaaact ctattattgt tgttgataaaggcatagatg caacttttag cttgagggtg 1260 tggactattc caatgagcca aaattattggggatcagaag gaagattact tttattaggt 1320 gacagaatat acatatatac tagatccacaagttggcaca gtaaattaca gttaggggta 1380 attgatattt ctgattataa taatataagaataaattgga cttggcataa tgtaccatca 1440 cggccaggaa atgatgaatg tccatggggtcattcatgcc cagacggatg tataacagga 1500 gtttacactg atgcatatcc gctaaacccatcggggagtg ttgtatcatc agtaattctt 1560 gactcacaaa agtctagaga aaacccaatcattacctact caacagctac aaatagaata 1620 aatgaattag ctatatataa cagaacacttccagctgcat atacaacaac aaattgtatc 1680 acacattatg ataaagggta ttgttttcatatagtagaaa taaatcacag aagtttgaat 1740 acgtttcaac ctatgttatt caaaacagaagttccaaaaa actgcagcta aatgatcatc 1800 gcatatcgga tgccagatga cattaaaagagaccaccaga cagacaacac aggagatgat 1860 gcaagatata aaggaataat 1880 74 900DNA IBRV unsure (812) Where n=unsure 74 gtttacaaat aataatataa cttttgataaaccgtatgca gcattgtatc catcagtagg 60 accaggaatc tattataagg gtaaagttatctttctcgga tatggaggtc tagagcatga 120 agaaaacgga gacgtaatat gtaatacaactggttgtcct ggcaaaacac agagagactg 180 taatcaggct tcttatagcc catggttctcaaataggaga atggtaaact ctattattgt 240 tgttgataaa ggcatagatg caacttttagcttgagggtg tggactattc caatgagcca 300 aaattattgg ggatcagaag gaagattacttttattaggt gacagaatat acatatatac 360 tagatccaca agttggcaca gtaaattacagttaggggta attgatattt ctgattataa 420 taatataaga ataaattgga cttggcataatgtaccatca cggccaggaa atgatgaatg 480 tccatggggt cattcatgcc cagacggatgtataacagga gtttacactg atgcatatcc 540 gctaaaccca tcggggagtg ttgtatcatcagtaattctt gactcacaaa agtctagaga 600 aaacccaatc attacctact caacagctacaaatagaata aatgaattag ctatatataa 660 cagaacactt ccagctgcat atacaacaacaaattgtatc acacattatg ataaagggta 720 ttgttttcat atagtagaaa taaacacagaagtttgaata cgtttcaacc tatgttattc 780 aaaacagaag ttccaaaaaa ctgcagctaaantgatcatc gcatatcgga tgccagatga 840 cattaaaaga gaccaccaga cagacaacacaggagatgat gcaagatata aaggaataat 900 75 198 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Linker 75 ccttatgtatcatacacata cgatttaggt gacactatag aatacaagct tgggctgcag 60 gtcgacgtccgcgctgctgc gccgcctggg ccggcggaag atctggtcat gctcgcggcc 120 gccatgcccccgggacacca tgtttttcct gccgcgcgcg gccgtcgact ctagaggatc 180 cccgggcgagctcgaatt 198 76 132 DNA Artificial Sequence Description of ArtificialSequence Synthetic Linker 76 ggatcccctc gacgtctggg gcgcgggggt ggtgctcttcgagacgctgg cctaccccaa 60 gacgatcaca cctttgcgca tctccacagc tcaacaatgaattccatgtt acgtcctgta 120 gaaaccccaa cc 132 77 132 DNA ArtificialSequence Description of Artificial Sequence Synthetic Linker 77cagggaggca aacaatgaat caacaactct cccgggagat gggggaggct aactgaaaca 60cggaaggtcg ccccccttaa gggtctcttg cacaatccag ccgcctccgt gttgctgcgt 120tcccggggat cc 132 78 122 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Linker 78 ctatagaata cacggaattc gagctcgcccgggtgagcgg cctaggccct cccccgaccg 60 cccgcgaccg aaaccccggg gatcctctagagtcgacctg cagcccaagc tctagcaacc 120 cc 122

What is claimed is:
 1. A recombinant infectious bovine rhinotracheitisvirus comprising a foreign DNA sequence inserted into an infectiousbovine rhinotracheitis viral genome, wherein the foreign DNA sequence isinserted within the BamHI C fragment of the infectious bovinerhinotracheitis viral genome and is capable of being expressed in aninfectious bovine rhinotracheitis virus infected host cell.
 2. Therecombinant infectious bovine rhinotracheitis virus of claim 1, whereinthe foreign DNA sequence is inserted within the largest BamHI-KpnIsubfragment of the BamHI C fragment of the infectious bovinerhinotracheitis viral genome.
 3. The recombinant infectious bovinerhinotracheitis virus of claim 2, wherein the foreign DNA sequence isinserted within a HindIII site located within the largest BamHI-KpnIsubfragment of the infectious bovine rhinotracheitis viral genome. 4.The recombinant infectious bovine rhinotracheitis virus of claim 2,wherein the foreign DNA sequence is inserted within an XbaI site locatedwithin the largest BamHI-KpnI subfragment of the infectious bovinerhinotracheitis viral genome.
 5. The recombinant infectious bovinerhinotracheitis virus of claim 1, wherein the foreign DNA sequence isinserted within the smallest KpnI-BamHI subfragment of the BamHI Cfragment of the infectious bovine rhinotracheitis viral genome.
 6. Therecombinant infectious bovine rhinotracheitis virus of claim 5, whereinthe foreign DNA sequence is inserted within an EcoRV site located withinthe smallest KpnI-BamHI subfragment of the infectious bovinerhinotracheitis viral genome.
 7. The recombinant infectious bovinerhinotracheitis virus of claim 1, further comprising a deletion in anon-essential region of the infectious bovine rhinotracheitis viralgenome.
 8. The recombinant infectious bovine rhinotracheitis virus ofclaim 7, wherein the deletion is in a US2 gene, a gG gene or a gE generegion.
 9. The recombinant infectious bovine rhinotracheitis virus ofclaim 1, wherein the foreign DNA sequence encodes a polypeptide.
 10. Therecombinant infectious bovine rhinotracheitis virus of claim 9, whereinthe polypeptide is antigenic in an animal into which the recombinantinfectious bovine rhinotracheitis virus is introduced.
 11. Therecombinant infectious bovine rhinotracheitis virus of claim 9, whereinthe polypeptide is E. coli beta-galactosidase.
 12. The recombinantinfectious bovine rhinotracheitis virus of claim 1, wherein the foreignDNA sequence encodes a cytokine or cytokine receptor.
 13. Therecombinant infectious bovine rhinotracheitis virus of claim 12, whereinthe cytokine or cytokine receptor is selected from the group consistingof: chicken myelomonocytic growth factor (cMGF), chicken interferon(cIFN), interleukin-2, interleukin-6, interleukin-12, interferons,granulocyte-macrophage colony stimulating factors, transforming growthfactor beta, epidermal growth factors, fibroblast growth factors,hepatocyte growth factor, insulin-like growth factors, B-nerve growthfactor, platelet-derived growth factor, vascular endothelial growthfactor, interleukin 1, IL-1 receptor antagonist, interleukin 3,interleukin 4, interleukin 5, IL-6 soluble receptor, interleukin 7,interleukin 8, interleukin 9, interleukin 10, interleukin 11,interleukin 13, angiogenin, chemokines, colony stimulating factors,erythropoietin, interferon gamma, leukemia inhibitory factor, oncostatinM, pleiotrophin, secretory leukocyte protease inhibitor, stem cellfactor, tumor necrosis factors, soluble TNF receptors and interleukinreceptors.
 14. The recombinant infectious bovine rhinotracheitis virusof claim 10, wherein the antigenic polypeptide is an equine influenzavirus neuroaminidase or an equine influenza virus hemagglutinin.
 15. Therecombinant infectious bovine rhinotracheiti virus of claim 10, whereinthe antigenic polypeptide is selected from the group consisting ofequine influenza virus type A/Alaska 91 neuraminidase, equine influenzavirus type A/Alaska 91 hemagglutinin, equine influenza virus typeA/Prague 56 neuraminidase, equine influenza virus type A/Prague 56hemagglutinin, equine influenza virus type A/Miami 63 neuraminidase,equine influenza virus type A/Miami 63 hemagglutinin, equine influenzavirus type A/Kentucky 81 neuraminidase equine influenza virus typeA/Kentucky 81 hemagglutinin, equine herpesvirus type 1 glycoprotein B,and equine herpesvirus type 1 glycoprotein D.
 16. The recombinantinfectious bovine rhinotracheitis virus of claim 10, wherein theantigenic polypeptide is selected from the group consisting of hogcholera virus gE1, hog cholera virus gE2, swine influenza virushemagglutinin, neuraminidase, matrix protein, nucleoprotein,pseudorabies virus gB, pseudorabies virus gC, pseudorabies virus gD, andPRRS virus ORF7.
 17. The recombinant infectious bovine rhinotracheitisvirus of claim 10, wherein the antigenic polypeptide is selected fromthe group consisting of: bovine respiratory syncytial virus attachmentprotein (BRSV G), bovine respiratory syncytial virus fusion protein(BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSVN), bovine coronavirus, bovine rotavirus glycoprotein 38, bovineparainfluenza virus type 3 fusion protein, and bovine parainfluenzavirus type 3 hemagglutinin neuraminidase.
 18. The recombinant infectiousbovine rhinotracheitis virus of claim 10, wherein the antigenicpolypeptide is bovine viral diarrhea virus (BVDV) glycoprotein 48 orbovine viral diarrhea virus (BVDV) glycoprotein
 53. 19. The recombinantinfectious bovine rhinotracheitis virus of claim 10, wherein theantigenic polypeptide is selected from the group consisting of Marek'sdisease virus gA, Marek's disease virus gB, Marek's disease virus gD,Newcastle disease virus HN, Newcastle disease virus F, infectiouslaryngotracheitis virus gB, infectious laryngotracheitis virus gI,infectious laryngotracheitis virus gD, infectious bursal disease virusVP2, infectious bursal disease virus VP3, infectious bursal diseasevirus VP4, infectious bursal disease virus polyprotein, infectiousbronchitis virus spike, infectious bronchitis virus matrix and chickanemia virus matrix.
 20. The recombinant infectious bovinerhinotracheitis virus of claim 1, wherein the foreign DNA sequence isunder the control of an endogenous infectious bovine rhinotracheitisvirus promoter.
 21. The recombinant infectious bovine rhinotracheitisvirus of claim 20, wherein the foreign DNA sequence is under the controlof a heterologous herpesvirus promoter.
 22. The recombinant infectiousbovine rhinotracheitis virus of claim 21, wherein the promoter isselected from the group consisting of a herpes simplex virus type 1(HSV-1) ICP4 protein promoter, an HSV-1 TK promoter, a pseudorabiesvirus (PRV) glycoprotein X promoter, a PRV gX promoter, an HCMVimmediate early promoter, a Marek's disease virus gA promoter, a Marek'sdisease virus gB promoter, a Marek's disease virus gD promoter, aninfectious laryngotracheitis virus gB promoter, a BHV-1.1 VP8 promoterand an infectious laryngotracheitis virus gD promoter.
 23. A vaccinewhich comprises an effective immunizing amount of the recombinantinfectious bovine rhinotracheitis virus of claim 1 and a carrier. 24.The vaccine of claim 23, wherein the carrier is a physiologicallybalanced culture medium containing stabilizing agents.
 25. The vaccineof claim 23, wherein the effective immunizing amount is from about 10³to about 10⁸ PFU/dose.
 26. The vaccine of claim 25, wherein theeffective immunizing amount is from about 10⁴ to about 10⁷ PFU/dose. 27.The vaccine of claim 25, wherein the effective immunizing amount is fromabout 10⁴ to about 10⁶ PFU/dose.
 28. A method of immunizing an animalagainst disease caused by infectious bovine rhinotracheitis virus whichcomprises administering to the animal an effective immunizing dose ofthe vaccine of claim
 23. 29. The recombinant infectious bovinerhinotracheitis virus of claim 10, wherein the antigenic polypeptide isfrom an organism or virus selected from the group consisting ofStreptococcus equi, equine infectious anemia virus, equine encephalitisvirus, equine rhinovirus and equine rotavirus.
 30. The recombinantinfectious bovine rhinotracheitis virus of claim 10, wherein theantigenic polypeptide is from an organism or virus selected from thegroup consisting of avian encephalomyelitis virus, avian reovirus, avianparamyxovirus, avian influenza virus, avian adenovirus, fowlpox virus,avian coronavirus, avian rotavirus, chick anemia virus, Salmonella spp.,E. coli, Pasteurella spp., Bordetella spp., Eimeria spp., Histomonasspp., Trichomonas spp., poultry nematodes, cestodes, trematodes, poultrymites, poultry lice and poultry protozoa.